Pretargeting methods and compounds

ABSTRACT

Methods, compounds, compositions and kits that relate to pretargeted delivery of diagnostic and therapeutic agents are disclosed. In particular, methods for radiometal labeling of biotin and for improved radiohalogenation of biotin, as well as related compounds, are described. Also, clearing agents, anti-ligand-targeting moiety conjugates, target cell retention enhancing moieties and additional methods are discussed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of prior application Ser. No.07/995,383, filed Dec. 23, 1992 now abandoned, which is a continuationin part of Ser. No. 07/895,588, filed Jun. 9, 1992, now U.S. Pat. No.5,283,342.

TECHNICAL FIELD

The present invention relates to methods, compounds, compositions andkits useful for delivering to a target site a targeting moiety that isconjugated to one member of a ligand/anti-ligand pair. Afterlocalization and clearance of the targeting moiety conjugate, direct orindirect binding of a diagnostic or therapeutic agent conjugate at thetarget site occurs. Methods for radiometal labeling of biotin and forimproved radiohalogenation of biotin, as well as the related compounds,are also disclosed. Also, clearing agents, anti-ligand targeting moietyconjugates, target cell-retention enhancing moieties and additionalmethods are set forth.

BACKGROUND OF THE INVENTION

Conventional cancer therapy is plagued by two problems. The generallyattainable targeting ratio (ratio of administered dose localizing totumor versus administered dose circulating in blood or ratio ofadministered dose localizing to tumor versus administered dose migratingto bone marrow) is low. Also, the absolute dose of radiation ortherapeutic agent delivered to the tumor is insufficient in many casesto elicit a significant tumor response. Improvement in targeting ratioor absolute dose to tumor is sought.

SUMMARY OF THE INVENTION

The present invention is directed to diagnostic and therapeuticpretargeting methods, moieties useful therein and methods of makingthose moieties. Such pretargeting methods are characterized by animproved targeting ratio or increased absolute dose to the target cellsites in comparison to conventional cancer therapy.

The present invention describes chelate-biotin compounds andradiohalogenated biotin compounds useful in diagnostic and therapeuticpretargeting methods. The present invention also provides targetingmoiety-ligand, such as biotin, compounds useful in diagnostic andtherapeutic pretargeting methods. Selection of moieties andmethodologies used to enhance internalization (of chemotherapeuticdrugs, for example) or to enhance retention at the target cell surface(of radionuclides, for example) is also discussed.

In addition, the present invention provides targetingmoiety-anti-ligand, such as avidin or streptavidin, compounds useful indiagnostic and therapeutic pretargeting methods. Preparation andpurification of such anti-ligand-targeting moiety compounds are alsodiscussed.

The present invention also provides clearing agents to facilitate theremoval of circulating targeting moiety-ligand (two-step) or targetingmoiety-anti-ligand (two-step) or anti-ligand (three-step) from themammalian recipient. Preferred clearing agents are classifiable asgalactose-based and non-galactose-based. Within each category,preferable clearing agents are polymeric or protein based.

Also, the present invention is directed to methods using streptavidin asan anti-ligand to enhance retention of radionuclide at target cellsites, with pretargeting protocols constituting one such method. Morespecifically, these embodiments of the present invention involve either(1) targeting moiety-streptavidin-radionuclide (with the radionuclidebound to streptavidin directly or through a chelate or linker), as wellas (2) targeting moiety-biotin administered prior tostreptavidin-radionuclide, or (3) biotin-radionuclide bound to apretargeted streptavidin containing molecule.

The present invention further provides pretargeting methods employingintraarterial administration of the therapeutic moiety-containingmolecule to achieve greater localization thereof to artery-suppliedtarget cell populations. Other methods of the present invention involvelo administration of short duration bone marrow protecting agents-priorto radionuclide-ligand molecule or radionuclide-anti-ligand moleculeadministration. Further, monovalent targeting moieties, such as Fv orFab antibody fragments, are useful in the inventive pretargetingmethods. Delivery of other, non-radioactive therapeutic agents, such aschemotherapeutic drugs, anti-tumor agents (e.g., cytokines) and toxins,to target cells using the pretargeting methods of the present inventionis also described.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates blood clearance of biotinylated antibody followingintravenous administration of avidin.

FIG. 2 depicts radiorhenium tumor uptake in a three-step pretargetingprotocol, as compared to administration of radiolabeled antibody(conventional means involving antibody that is covalently linked tochelated radiorhenium).

FIG. 3 depicts the tumor uptake profile of NR-LU-10-streptavidinconjugate (LU-10-StrAv) in comparison to a control profile of nativeNR-LU-10 whole antibody.

FIG. 4 depicts the tumor uptake and blood clearance profiles ofNR-LU-10-streptavidin conjugate.

FIG. 5 depicts the rapid clearance from the blood of asialoorosomucoidin comparison with orosomucoid in terms of percent injected dose ofI-125-labeled protein.

FIG. 6 depicts the 5 minute limited biodistribution of asialoorosomucoidin comparison with orosomucoid in terms of percent injected dose ofI-125-labeled protein.

FIG. 7 depicts NR-LU-10-streptavidin conjugate blood clearance uponadministration of three controls (∘, , ▪) and two doses of a clearingagent (, □) at 25 hours post-conjugate administration.

FIG. 8 shows limited biodistribution data for LU-10-StrAv conjugate uponadministration of three controls (Groups 1, 2 and 5) and two doses ofclearing agent (Groups 3 and 4) at two hours post-clearing agentadministration.

FIG. 9 depicts NR-LU-10-streptavidin conjugate serum biotin bindingcapability at 2 hours post-clearing agent administration.

FIG. 10 depicts NR-LU-10-streptavidin conjugate blood clearance overtime upon administration of a control (∘) and three doses of a clearingagent (∇, Δ, □) at 24 hours post-conjugate administration.

FIG. 11A depicts the blood clearance of LU-10-StrAv conjugate uponadministration of a control (PBS) and three doses (50, 20 and 10 μg) ofclearing agent at two hours post-clearing agent administration.

FIG. 11B depicts LU-10-StrAv conjugate serum biotin binding capabilityupon administration of a control (PBS) and three doses (50, 20 and 10μg) of clearing agent at two hours post-clearing agent administration.

FIG. 12 depicts the prolonged tumor retention of NR-LU-10-streptavidinconjugate (▴) relative to NR-LU-10 whole antibody (Δ) over time.

FIG. 13 depicts the prolonged liver retention of a pre-formed complex ofNR-LU-10-biotin (∘; chloramine T labeled with I-125) complexed withstreptavidin (; PIP-I-131 labeled).

FIG. 14 depicts the prolonged liver retention of Biotin-PIP-I-131 labelrelative to the streptavidin-NR-LU-10-(PIP-I-125) label.

FIG. 15A depicts tumor uptake for increasing doses of PIP-Biocytin interms of %ID/G.

FIG. 15B depicts tumor uptake for increasing doses of PIP-Biocytin overtime in terms of pMOL/G.

FIG. 16A depicts tumor versus blood localization of a 0.5 μg dose ofPIP-Biocytin over time in terms of %ID/G.

FIG. 16B depicts tumor versus blood localization of a 0.5 μg dose ofPIP-Biocytin in terms of %ID.

FIG. 17A depicts tumor uptake of LU-10-StrAv and PIP-Biocytin over timein terms of %ID/G.

FIG. 17B depicts blood clearance of LU-10-StrAv and PIP-Biocytin overtime in terms of %ID/G.

FIG. 18 depicts PIP-Biocytin:LU-10-StrAv ratio in tumor and blood overtime.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to set forthdefinitions of certain terms to be used within the disclosure.

Targeting moiety: A molecule that binds to a defined population ofcells. The targeting moiety may bind a receptor, an oligonucleotide, anenzymatic substrate, an antigenic determinant, or other binding sitepresent on or in the target cell population. Antibody is used throughoutthe specification as a prototypical example of a targeting moiety. Tumoris used as a prototypical example of a target in describing the presentinvention.

Ligand/anti-ligand pair: A complementary/anti-complementary set ofmolecules that demonstrate specific binding, generally of relativelyhigh affinity. Exemplary ligand/anti-ligand pairs includehapten/antibody, lectin/carbohydrate, ligand/receptor, andbiotin/avidin. Biotin/avidin is used throughout the specification as aprototypical example of a ligand/anti-ligand pair.

Anti-ligand: As defined herein, an "anti-ligand" demonstrates highaffinity, and preferably, multivalent binding of the complementaryligand. Preferably, the anti-ligand is large enough to avoid rapid renalclearance, and contains sufficient multivalency to accomplishcrosslinking and aggregation of targeting moiety-ligand conjugates.Univalent anti-ligands are also contemplated by the present invention.Anti-ligands of the present invention may exhibit or be derivitized toexhibit structural features that direct the uptake thereof, e.g.,galactose residues that direct liver uptake. Avidin and streptavidin areused herein as prototypical anti-ligands.

Avidin: As defined herein, "avidin" includes avidin, streptavidin andderivatives and analogs thereof that are capable of high affinity,multivalent or univalent binding of biotin.

Ligand: As defined herein, a "ligand" is a relatively small, solublemolecule that exhibits rapid serum, blood and/or whole body clearancewhen administered intravenously in an animal or human. Biotin is used asthe prototypical ligand.

Active Agent: A diagnostic or therapeutic agent ("the payload"),including radionuclides, drugs, anti-tumor agents, toxins and the like.Radionuclide therapeutic agents are used as prototypical active agents.

N_(x) S_(y) Chelates: As defined herein, the term "N_(x) S_(y) chelates"includes bifunctional chelators that are capable of (i) coordinatelybinding a metal or radiometal and (ii) covalently attaching to atargeting moiety, ligand or anti-ligand. Particularly preferred N_(x)S_(y) chelates have N₂ S₂ and N₃ S cores. Exemplary N_(x) S_(y) chelatesare described in Fritzberg et al., Proc. Natl. Acad. Sci. USA85:4024-29, 1988; in Weber et al., Bioconj. Chem. 1:431-37, 1990; and inthe references cited therein, for instance.

Pretargeting: As defined herein, pretargeting involves target sitelocalization of a targeting moiety that is conjugated with one member ofa ligand/anti-ligand pair; after a time period sufficient for optimaltarget-to-non-target accumulation of this targeting moiety conjugate,active agent conjugated to the opposite member of the ligand/anti-ligandpair is administered and is bound (directly or indirectly) to thetargeting moiety conjugate at the target site. Three-step and otherrelated methods described herein are also encompassed.

Clearing Agent: An agent capable of binding, complexing or otherwiseassociating with an administered moiety (e.g., targeting moiety-ligand,targeting moiety-anti-ligand or anti-ligand alone) present in therecipient's circulation, thereby facilitating circulating moietyclearance from the recipient's body, removal from blood circulation, orinactivation thereof in circulation. The clearing agent is preferablycharacterized by physical properties, such as size, charge,configuration or a combination thereof, that limit clearing agent accessto the population of target cells recognized by a targeting moiety usedin the same treatment protocol as the clearing agent.

Target Cell Retention: The amount of time that a radionuclide or othertherapeutic agent remains at the target cell surface or within thetarget cell. Catabolism of conjugates or molecules containing suchtherapeutic agents appears to be primarily responsible for the loss oftarget cell retention.

A recognized disadvantage associated with in vivo administration oftargeting moiety-radioisotopic conjugates for imaging or therapy islocalization of the attached radioactive agent at both non-target andtarget sites. Until the administered radiolabeled conjugate clears fromthe circulation, normal organs and tissues are transitorily exposed tothe attached radioactive agent. For instance, radiolabeled wholeantibodies that are administered in vivo exhibit relatively slow bloodclearance; maximum target site localization generally occurs 1-3 dayspost-administration. Generally, the longer the clearance time of theconjugate from the circulation, the greater the radioexposure ofnon-target organs.

These characteristics are particularly problematic with humanradioimmunotherapy. In human clinical trials, the long circulatinghalf-life of radioisotope bound to whole antibody causes relativelylarge doses of radiation to be delivered to the whole body. Inparticular, the bone marrow, which is very radiosensitive; is thedose-limiting organ of non-specific toxicity.

In order to decrease radioisotope exposure of non-target tissue,potential targeting moieties generally have been screened to identifythose that display minimal non-target reactivity, while retaining targetspecificity and reactivity. By reducing non-target exposure (and adversenon-target localization and/or toxicity) increased doses of aradiotherapeutic conjugate may be administered; moreover, decreasednon-target accumulation of a radiodiagnostic conjugate leads to improvedcontrast between background and target.

Therapeutic drugs, administered alone or as targeted conjugates, areaccompanied by similar disadvantages. Again, the goal is administrationof the highest possible concentration of drug (to maximize exposure oftarget tissue), while remaining below the threshold of unacceptablenormal organ toxicity (due to non-target tissue exposure). Unlikeradioisotopes, however, therapeutic drugs need to be taken into a targetcell to exert a cytotoxic effect. In the case of targetingmoiety-therapeutic drug conjugates, it would be advantageous to combinethe relative target specificity of a targeting moiety with a means forenhanced target cell internalization of the targeting moiety-drugconjugate.

In contrast, enhanced target cell internalization is disadvantageous ifone administers diagnostic agent-targeting moiety conjugates.Internalization of diagnostic conjugates results in cellular catabolismand degradation of the conjugate. Upon degradation, small adducts of thediagnostic agent or the diagnostic agent per se may be released from thecell, thus eliminating the ability to detect the conjugate in atarget-specific manner.

One method for reducing non-target tissue exposure to a diagnostic ortherapeutic agent involves "pretargeting" the targeting moiety at atarget site, and then subsequently administering a rapidly clearingdiagnostic or therapeutic agent conjugate that is capable of binding tothe "pretargeted" targeting moiety at the target site. A description ofsome embodiments of the pretargeting technique may be found in U.S. Pat.No. 4,863,713 (Goodwin et al.).

A typical pretargeting approach ("three-step") is schematically depictedbelow. ##STR1## Briefly, this three-step pretargeting protocol featuresadministration of an antibody-ligand conjugate, which is allowed tolocalize at a target site and to dilute in the circulation. Subsequentlyadministered anti-ligand binds to the antibody-ligand conjugate andclears unbound antibody-ligand conjugate from the blood. Preferredanti-ligands are large and contain sufficient multivalency to accomplishcrosslinking and aggregation of circulating antibody-ligand conjugates.The clearing by anti-ligand is probably attributable to anti-ligandcrosslinking and/or aggregation of antibody-ligand conjugates that arecirculating in the blood, which leads to complex/aggregate clearance bythe recipient's RES (reticuloendothelial system). Anti-ligand clearanceof this type is preferably accomplished with a multivalent molecule;however, a univalent molecule of sufficient size to be cleared by theRES on its own could also be employed. Alternatively, receptor-basedclearance mechanisms, e.g., Ashwell receptor galactose residuerecognition mechanisms, may be responsible for anti-ligand clearance.Such clearance mechanisms are less dependent upon the valency of theanti-ligand with respect to the ligand than the RES complex/aggregateclearance mechanisms. It is preferred that the ligand-anti-ligand pairdisplays relatively high affinity binding.

A diagnostic or therapeutic agent-ligand conjugate that exhibits rapidwhole body clearance is then administered. When the circulation bringsthe active agent-ligand conjugate in proximity to the target cell-boundantibody-ligand-anti-ligand complex, anti-ligand binds the circulatingactive agent-ligand conjugate and produces anantibody-ligand:anti-ligand:ligand-active agent "sandwich" at the targetsite. Because the diagnostic or therapeutic agent is attached to arapidly clearing ligand (rather than antibody, antibody fragment orother slowly clearing targeting moiety), this technique promisesdecreased non-target exposure to the active agent.

Alternate pretargeting methods eliminate the step of parenterallyadministering an anti-ligand clearing agent. These "two-step" proceduresfeature targeting moiety-ligand or targeting moiety-anti-ligandadministration, followed by administration of active agent conjugated tothe opposite member of the ligand-anti-ligand pair. As an optional step"1.5" in the two-step pretargeting methods of the present invention, aclearing agent (preferably other than ligand or anti-ligand) isadministered to facilitate the clearance of circulating targetingmoiety-containing conjugate.

In the two-step pretargeting approach, the clearing agent preferablydoes not become bound to the target cell population, either directly orthrough the previously administered and target cell bound targetingmoiety-anti-ligand or targeting moiety-ligand conjugate. An example oftwo-step pretargeting involves the use of biotinylated human transferrinas a clearing agent for avidin-targeting moiety conjugate, wherein thesize of the clearing agent results in liver clearance oftransferrin-biotin-circulating avidin-targeting moiety complexes andsubstantially precludes association with the avidin-targeting moietyconjugates bound at target cell sites. (See, Goodwin, D. A., Antibod.Immunoconj. Radiopharm., 4: 427-34, 1991).

The two-step pretargeting approach overcomes certain disadvantagesassociated with the use of a clearing agent in a three-step pretargetedprotocol. More specifically, data obtained in animal models demonstratethat in vivo anti-ligand binding to a pretargeted targetingmoiety-ligand conjugate (i.e., the cell-bound conjugate) removes thetargeting moiety-ligand conjugate from the target cell. One explanationfor the observed phenomenon is that the multivalent anti-ligandcrosslinks targeting moiety-ligand conjugates on the cell surface,thereby initiating or facilitating internalization of the resultantcomplex. The apparent loss of targeting moiety-ligand from the cellmight result from internal degradation of the conjugate and/or releaseof active agent from the conjugate (either at the cell surface orintracellularly). An alternative explanation for the observed phenomenonis that permeability changes in the target cell's membrane allowincreased passive diffusion of any molecule into the target cell. Also,some loss of targeting moiety-ligand may result from alteration in theaffinity by subsequent binding of another moiety to the targetingmoiety-ligand, e.g., anti-idiotype monoclonal antibody binding causesremoval of tumor bound monoclonal antibody.

The present invention recognizes that this phenomenon (apparent loss ofthe targeting moiety-ligand from the target cell) may be used toadvantage with regard to in vivo delivery of therapeutic agentsgenerally, or to drug delivery in particular. For instance, a targetingmoiety may be covalently linked to both ligand and therapeutic agent andadministered to a recipient. Subsequent administration of anti-ligandcrosslinks targeting moiety-ligand-therapeutic agent tripartiteconjugates bound at the surface, inducing internalization of thetripartite conjugate (and thus the active agent). Alternatively,targeting moiety-ligand may be delivered to the target cell surface,followed by administration of anti-ligand-therapeutic agent.

In one aspect of the present invention, a targeting moiety-anti-ligandconjugate is administered in vivo; upon target localization of thetargeting moiety-anti-ligand conjugate (i.e., and clearance of thisconjugate from the circulation), an active agent-ligand conjugate isparenterally administered. This method enhances retention of thetargeting moiety-anti-ligand:ligand-active agent complex at the targetcell (as compared with targeting moiety-ligand:anti-ligand:ligand-activeagent complexes and targeting moiety-ligand:anti-ligand-active agentcomplexes). Although a variety of ligand/anti-ligand pairs may besuitable for use within the claimed invention, a preferredligand/anti-ligand pair is biotin/avidin.

In a second aspect of the invention, radioiodinated biotin and relatedmethods are disclosed. Previously, radioiodinated biotin derivativeswere of high molecular weight and were difficult to characterize. Theradioiodinated biotin described herein is a low molecular weightcompound that has been easily and well characterized.

In a third aspect of the invention, a targeting moiety-ligand conjugateis administered in vivo; upon target localization of the targetingmoiety-ligand conjugate (i.e., and clearance of this conjugate from thecirculation), a drug-anti-ligand conjugate is parenterally administered.This two-step method not only provides pretargeting of the targetingmoiety conjugate, but also induces internalization of the subsequenttargeting moiety-ligand-anti-ligand-drug complex within the target cell.Alternatively, another embodiment provides a three-step protocol thatproduces a targeting moiety-ligand:anti-ligand:ligand-drug complex atthe surface, wherein the ligand-drug conjugate is administeredsimultaneously or within a short period of time after administration ofanti-ligand (i.e., before the targeting moiety-ligand-anti-ligandcomplex has been removed from the target cell surface).

In a fourth aspect of the invention, methods for radiolabeling biotinwith technetium-99m, rhenium-186 and rhenium-188 are disclosed.Previously, biotin derivatives were radiolabeled with indium-111 for usein pretargeted immunoscintigraphy (for instance, Virzi et al., Nucl.Med. Biol. 18:719-26, 1991; Kalofonos et al., J. Nucl. Med. 31: 1791-96,1990; Paganelli et al. Canc. Res. 51:5960-66, 1991). However, ^(99m) Tcis a particularly preferred radionuclide for immunoscintigraphy due to(i) low cost, (ii) convenient supply and (iii) favorable nuclearproperties. Rhenium-186 displays chelating chemistry very similar to^(99m) Tc, and is considered to be an excellent therapeutic radionuclide(i.e., a 3.7 day half-life and 1.07 MeV maximum particle that is similarto ¹³¹ I). Therefore, the claimed methods for technetium and rheniumradiolabeling of biotin provide numerous advantages.

The "targeting moiety" of the present invention binds to a definedtarget cell population, such as tumor cells. Preferred targetingmoieties useful in this regard include antibody and antibody fragments,peptides, and hormones. Proteins corresponding to known cell surfacereceptors (including low density lipoproteins; transferrin and insulin),fibrinolytic enzymes, anti-HER2, and biological response modifiers(including interleukin, interferon, erythropoietin andcolony-stimulating factor) are also preferred targeting moieties. Also,anti-EGF receptor antibodies, which internalize following binding to thereceptor and traffic to the nucleus to an extent, are preferredtargeting moieties for use in the present invention to facilitatedelivery of Auger emitters and nucleus binding drugs to target cellnuclei. Oligonucleotides, e.g., antisense oligonucleotides that arecomplementary to portions of target cell nucleic acids (DNA or RNA), arealso useful as targeting moieties in the practice of the presentinvention. Oligonucleotides binding to cell surfaces are also useful.Analogs of the above-listed targeting moieties that retain the capacityto bind to a defined target cell population may also be used within theclaimed invention. In addition, synthetic targeting moieties may bedesigned.

Functional equivalents of the aforementioned molecules are also usefulas targeting moieties of the present invention. One targeting moietyfunctional equivalent is a "mimetic" compound, an organic chemicalconstruct designed to mimic the proper configuration and/or orientationfor targeting moiety-target cell binding. Another targeting moietyfunctional equivalent is a short polypeptide designated as a "minimal"polypeptide, constructed using computer-assisted molecular modeling andmutants having altered binding affinity, which minimal polypeptidesexhibit the binding affinity of the targeting moiety.

Preferred targeting moieties of the present invention are antibodies(polyclonal or monoclonal), peptides, oligonucleotides or the like.Polyclonal antibodies useful in the practice of the present inventionare polyclonal (Vial and Callahan, Univ. Mich. Med. Bull., 20: 284-6,1956), affinity-purified polyclonal or fragments thereof (Chao et al.,Res. Comm. in Chem. Path. & Pharm., 9: 749-61, 1974).

Monoclonal antibodies useful in the practice of the present inventioninclude whole antibody and fragments thereof. Such monoclonal antibodiesand fragments are producible in accordance with conventional techniques,such as hybridoma synthesis, recombinant DNA techniques and proteinsynthesis. Useful monoclonal antibodies and fragments may be derivedfrom any species (including humans) or may be formed as chimericproteins which employ sequences from more than one species. See,generally, Kohler and Milstein, Nature, 256: 495-97, 1975, Eur. J.Immunol., 6: 511-19, 1976.

Human monoclonal antibodies or "humanized" murine antibody are alsouseful as targeting moieties in accordance with the present invention.For example, murine monoclonal antibody may be "humanized" bygenetically recombining the nucleotide sequence encoding the murine Fvregion (i.e., containing the antigen binding sites) or thecomplementarity determining regions thereof with the nucleotide sequenceencoding a human constant domain region and an Fc region, e.g., in amanner similar to that disclosed in European Patent Application No.0,411,893 A2. Humanized targeting moieties are recognized to decreasethe immunoreactivity of the antibody or polypeptide in the hostrecipient, permitting an increase in the half-life and a reduction inthe possibility of adverse immune reactions.

Types of active agents (diagnostic or therapeutic) useful herein includetoxins, anti-tumor agents, drugs and radionuclides. Several of thepotent toxins useful within the present invention consist of an A and aB chain. The A chain is the cytotoxic portion and the B chain is thereceptor-binding portion of the intact toxin molecule (holotoxin).Because toxin B chain may mediate non-target cell binding, it is oftenadvantageous to conjugate only the toxin A chain to a targeting protein.However, while elimination of the toxin B chain decreases non-specificcytotoxicity, it also generally leads to decreased potency of the toxinA chain-targeting protein conjugate, as compared to the correspondingholotoxin-targeting protein conjugate.

Preferred toxins in this regard include holotoxins, such as abrin,ricin, modeccin, Pseudomonas exotoxin A, Diphtheria toxin, pertussistoxin and Shiga toxin; and A chain or "A chain-like" molecules, such asricin A chain, abrin A chain, modeccin A chain, the enzymatic portion ofPseudomonas exotoxin A, Diphtheria toxin A chain, the enzymatic portionof pertussis toxin, the enzymatic portion of Shiga toxin, gelonin,pokeweed antiviral protein, saporin, tritin, barley toxin and snakevenom peptides. Ribosomal inactivating proteins (RIPs), naturallyoccurring protein synthesis inhibitors that lack translocating andcell-binding ability, are also suitable for use herein.

Preferred drugs suitable for use herein include conventionalchemotherapeutics, such as vinblastine, doxorubicine bleomycin,methotrexate, 5-fluorouracil, 6-thioguanine, cytarabine,cyclophosphamide and cis-platinum, as well as other conventionalchemotherapeutics as described in Cancer: Principles and Practice ofOncology, 2d ed., V. T. DeVita, Jr., S. Hellman, S. A. Rosenberg, J. B.Lippincott Co., Philadelphia, Pa., 1985, Chapter 14. A particularlypreferred drug within the present invention is a trichothecene.

Experimental drugs, such as mercaptopurine, N-methylformamide,2-amino-1,3,4-thiadiazole, melphalan, hexamethylmelamine, galliumnitrate, 3% thymidine, dichloromethotrexate, mitoguazone, suramin,bromodeoxyuridine, iododeoxyuridine, semustine,1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea,N,N'-hexamethylene-bis-acetamide, azacitidine, dibromodulcitol, Erwiniaasparaginase, ifosfamide, 2-mercaptoethane sulfonate, teniposide, taxol,3-deazauridine, soluble Baker's antifol, homoharringtonine,cyclocytidine, acivicin, ICRF-187, spiromustine, levamisole,chlorozotocin, aziridinyl benzoquinone, spirogermanium, aclarubicin,pentostatin, PALA, carboplatin, amsacrine, caracemide, iproplatin,misonidazole, dihydro-5-azacytidine, 4'-deoxy-doxorubicin, menogaril,triciribine phosphate, fazarabine, tiazofurin, teroxirone, ethiofos,N-(2-hydroxyethyl)-2-nitro-1H-imidazole-1-acetamide, mitoxantrone,acodazole, amonafide, fludarabine phosphate, pibenzimol, didemnin B,merbarone, dihydrolenperone; flavone-8-acetic acid, oxantrazole,ipomeanol, trimetrexate, deoxyspergualin, echinomycin, anddideoxycytidine (see NCI Investigational Drugs, Pharmaceutical Data1987, NIH Publication No. 88-2141, Revised November 1987) are alsopreferred.

Radionuclides useful within the present invention includegamma-emitters, positron-emitters, Auger electron-emitters, X-rayemitters and fluorescence-emitters, with beta- or alpha-emitterspreferred for therapeutic use. Radionuclides are well-known in the artand include ¹²³ I, ¹²⁵ I, ¹³⁰ I, ¹³¹ I, ¹³³ I, ¹³⁵ I, ⁴⁷ SC, ⁷² As, ⁷²Se, ⁹⁰ Y, ⁹⁷ Ru, ¹⁰⁰ Pd, ^(101m) Rh, ¹¹⁹ Sb, ¹²⁸ Ba, ¹⁹⁷ Hg, ²¹¹ At, ²¹²Bi, ¹⁵³ Sm, ¹⁶⁹ Eu, ²¹² Pb, ¹⁰⁹ Pd, ¹¹¹ In, ⁶⁷ Ga, ⁶⁸ Ga, ⁶⁷ Cu, ⁷⁵ Br,⁷⁶ Br, ⁷⁷ Br, ^(99m) Tc, ¹¹ C, ¹³ N, ¹⁵ O and ¹⁸ F. Preferredtherapeutic radionuclides include ¹⁸⁸ Re, ¹⁸⁶ Re, ²⁰³ Pb, ²¹² Pb, ²¹²Bi, ¹⁰⁹ Pd, ⁶⁴ Cu, ⁶⁷ Cu, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ⁷⁷ Br, ²¹¹ At, ⁹⁷ Ru, ¹⁰⁵Rh, ¹⁹⁸ Au and ¹⁹⁹ Ag or ¹⁷⁷ Lu.

Other anti-tumor agents are administrable in accordance with the presentinvention. Exemplary anti-tumor agents include cytokines, such as IL-2,tumor necrosis factor or the like, lectin inflammatory responsepromoters (selecting), such as L-selectin, E-selectin, P-selectin or thelike, and like molecules.

Ligands suitable for use within the present invention include biotin,haptens, lectins, epitopes and analogs and derivatives thereof. Usefulcomplementary anti-ligands include avidin (for biotin), carbohydrates(for lectins) and antibody, fragments or analogs thereof, includingmimetics (for haptens and epitopes). Preferred ligands and anti-ligandsbind to each other with an affinity of at least about k_(D) ≧10⁹ M.

One component to be administered in a preferred two-step pretargetingprotocol is a targeting moiety-avidin or a targeting moiety-streptavidinconjugate. The preferred targeting moiety useful in these embodiments ofthe present invention is a monoclonal antibody. Protein-proteinconjugations are generally problematic due to the formation ofundesirable byproducts, including high molecular weight and cross linkedspecies, however. A non-covalent synthesis technique involving reactionof biotinylated antibody with streptavidin has been reported to resultin substantial byproduct formation. Also, at least one of the fourbiotin binding sites on the streptavidin is used to link the antibodyand streptavidin, while another such binding site may be stericallyunavailable for biotin binding due to the configuration of thestreptavidin-antibody conjugate.

Thus, covalent streptavidin-antibody conjugation is preferred, but highmolecular weight byproducts are often obtained. The degree ofcrosslinking and aggregate formation is dependent upon several factors,including the level of protein derivitization using heterobifunctionalcrosslinking reagents. Sheldon et al., Appl. Radiat. Isot. 43:1399-1402, 1992, discuss preparation of covalent thioether conjugates byreacting succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC)derivitized antibody and iminothiolane-derivitized streptavidin.

Streptavidin-proteinaceous targeting moiety conjugates are preferablyprepared as described in Example XI below, with the preparationinvolving the steps of: preparation of SMCC-derivitized streptavidin;preparation of DTT-reduced proteinaceous targeting moiety; conjugationof the two prepared moieties; and purification of the monosubstitutedconjugate. The purified fraction is preferably further characterized byone or more of the following techniques: HPLC size exclusion, SDS-PAGE,immunoreactivity, biotin binding capacity and in vivo studies.

Alternatively, thioether conjugates useful in the practice of thepresent invention may be formed using other thiolating agents, such asSPDP, iminothiolate, SATA or the like, or other thio-reactiveheterobifunctional cross linkers, such asm-maleimidobenzoyl-N-hydroxysuccinimide ester,N-succinimidyl(4-iodoacetyl)aminobenzoate or the like.

Streptavidin-proteinaceous targeting moiety conjugates of the presentinvention can also be formed by conjugation of a lysine epsilon aminogroup of one protein with a maleimide-derivitized form of the otherprotein. For example, at pH 8-10, lysine epsilon amino moieties reactwith protein maleimides, prepared, for instances by treatment of theprotein with SMCC, to generate stable amine covalent conjugates. Inaddition, conjugates can be prepared by reaction of lysine epsilon aminomoieties of one protein with aldehyde functionalities of the otherprotein. The resultant imine bond is reducible to generate thecorresponding stable amine bond. Aldehyde functionalities may begenerated, for example, by oxidation of protein sugar residues or byreaction with aldehyde-containing heterobifunctional cross linkers.

Another method of forming streptavidin-targeting moiety conjugatesinvolves immobilized iminobiotin that binds SMCC-derivitizedstreptavidin. In this conjugation/purification method, the reversiblebinding character of iminobiotin (immobilized) to streptavidin isexploited to readily separate conjugate from the unreacted targetingmoiety. Iminobiotin binding can be reversed under conditions of lower pHand elevated ionic strength, e.g., NH₂ OAc, pH 4 (50 mM) with 0.5 MNaCl.

For streptavidin, for example, the conjugation/purification proceeds asfollows:

SMCC-derivitized streptavidin is bound to immobilized iminobiotin(Pierce Chemical Co., St. Louis, Mo.), preferably in column format;

a molar excess (with respect to streptavidin) of DTT-reduced antibody(preferably free of reductant) is added to the nitrogen-purged,phosphate-buffered iminobiotin column wherein the SMCC-streptavidin isbound (DTT-reduced antibody will saturate the bound SMCC-streptavidin,and unbound reduced antibody passing through the column can be reused);

the column is washed free of excess antibody; and

a buffer that lowers the pH and increases ionic strength is added to thecolumn to elute streptavidin-antibody conjugate in pure form.

As indicated above, targeting moiety-mediated ligand-anti-ligandpretargeting involves the localization of either targeting moiety-ligandor targeting moiety-anti-ligand at target tissue. Often, peak uptake tosuch target tissue is achieved before the circulating level of targetingmoiety-containing conjugate in the blood is sufficiently low to permitthe attainment of an optimal target-to-non-target conjugate ratio. Toobviate this problem, two approaches are useful. The first approachallows the targeting moiety-containing conjugate to clear from the bloodby "natural" or endogenous clearance mechanisms. This method iscomplicated by variations in systemic clearance of-proteins and byendogenous ligand or anti-ligand. For example, endogenous biotin mayinterfere with the preservation of biotin binding sites on astreptavidin-targeting moiety conjugate.

The second approach for improving targeting moiety-ligand or targetingmoiety-anti-ligand conjugate target-to-blood ratio "chases" theconjugate from the circulation through in vivo complexation of conjugatewith a molecule constituting or containing the complementary anti-ligandor ligand. When biotinylated antibodies are used as a ligand-targetingmoiety conjugate, for example, avidin forms relatively large aggregatedspecies upon complexation with the circulating biotinylated antibody,which aggregated species are rapidly cleared from the blood by the RESuptake. See, for example, U.S. Pat. No. 4,863,713. One problem with thismethod, however, is the potential for cross-linking and internalizingtumor-bound biotinylated antibody by avidin.

When avidin-targeting moiety conjugates are employed, poly-biotinylatedtransferrin has been used to form relatively large aggregated speciesthat are cleared by RES uptake. See, for example, Goodwin, J. Nucl. Med.33(10):1816-18, 1992). Poly-biotinylated transferrin also has thepotential for cross-linking and internalizing tumor-boundavidinylated-targeting moiety; however. In addition, both "chase"methodologies involve the prolonged presence of aggregated moieties ofintermediate, rather than large, size (which are not cleared as quicklyas large size particles by RES uptake), thereby resulting in serumretention of subsequently administered ligand-active agent oranti-ligand active agent. Such serum retention unfavorably impacts thetarget cell-to-blood targeting ratio.

The present invention provides clearing agents of protein andnon-protein composition having physical properties facilitating use forin vivo complexation and blood clearance of anti-ligand/ligand (e.g.,avidin/biotin)-targeting moiety (e.g., antibody) conjugates. Theseclearing agents are useful in improving the target:blood ratio oftargeting moiety conjugate. Other applications of these clearing agentsinclude lesional imaging or therapy involving blood clots and the like,employing antibody-active agent delivery modalities. For example,efficacious anti-clotting agent provides rapid target localization andhigh target:non-target targeting ratio. Active agents administered inpretargeting protocols of the present invention using efficient clearingagents are targeted in the desirable manner and are, therefore, usefulin the imaging/therapy of conditions such as pulmonary embolism and deepvein thrombosis.

Clearing agents useful in the practice of the present inventionpreferably exhibit one or more of the following characteristics:

rapid, efficient complexation with targeting moiety-ligand (oranti-ligand) conjugate in vivo;

rapid clearance from the blood of targeting moiety conjugate capable ofbinding a subsequently administered complementary anti-ligand or ligandcontaining molecule;

high capacity for clearing (or inactivating) large amounts of targetingmoiety conjugate; and

low immunogenicity.

Preferred clearing agents include galactose-based and non-galactosebased molecules. Protein-type galactose-based clearing agents includeproteins having endogenous exposed galactose residues or which have beenderivitized to expose or incorporate such galactose residues. Exposedgalactose residues direct the clearing agent to rapid clearance byendocytosis into the liver through specific receptors therefor (Ashwellreceptors). These receptors bind the clearing agent, and induceendocytosis into the hepatocyte, leading to fusion with a lysosome andrecycle of the receptor back to the cell surface. This clearancemechanism is characterized by high efficiency, high capacity and rapidkinetics.

An exemplary clearing agent of the protein-based/galactose-bearingvariety is the asialoorosomucoid derivative of human alpha-1acidglycoprotein (orosomucoid, molecular weight=41,000 Dal; isoelectricpoint=1.8-2.7). The rapid clearance from the blood of asialoorosomucoidhas been documented by Galli, et al., J. of Nucl. Med. Allied Sci.32(2): 110-16, 1988.

Treatment of orosomucoid with neuraminidase removes sialic acidresidues, thereby exposing galactose residues. Other such derivitizedclearing agents include, for example, galactosylated albumin,galactosylated-IgM, galactosylated-IgG, asialohaptoglobin, asialofetuin,asialoceruloplasmin and the like.

In addition, clearing agents based upon human proteins, especially humanserum proteins, are less immunogenic upon administration into the serumof a human recipient. Another advantage of using asialoorosomucoid isthat human orosomucoid is commercially available from, for example,Sigma Chemical Co, St. Louis,, Mo.

One way to prevent clearing agent compromise of target-bound conjugatethrough direct complexation is through use of a clearing agent of a sizesufficient to render the clearing agent less capable of diffusion intothe extravascular space and binding to target-associated conjugate. Thisstrategy is useful alone or in combination with the aforementionedrecognition that exposed galactose residues direct rapid liver uptake.This size-exclusion strategy enhances the effectiveness ofnon-galactose-based clearing agents of the present invention. Thecombination (exposed galactose and size) strategy improves theeffectiveness of "protein-type" or "polymer-type" galactose-basedclearing agents.

Galactose-based clearing agents include galactosylated, biotinylatedproteins (to remove circulating streptavidin-targeting moietyconjugates, for example) of intermediate molecular weight (ranging fromabout 40,000 to about 200,000 Dal), such as asialoorosomucoid,galactosyl-biotinyl-human serum albumin or other galactosylated andbiotinylated derivatives of non-immunogenic soluble-natural proteins, aswell as biotin- and galactose-derivitized polyglutamate, polylysine,polyarginine, polyaspartate and the like. High molecular weight moieties(ranging from about 200,000 to about 1,000,000 Dal) characterized bypoor target access, including galactosyl-biotinyl-IgM or -IgG(approximately 150,000 Dal) molecules, as well as galactose- andbiotin-derivitized transferrin conjugates of human serum albumin, IgGand IgM molecules and the like, can also be used as clearing agents ofthe claimed invention. Chemically modified polymers of intermediate orhigh molecular weight (ranging from about 40,000 to about 1,000,000Dal), such as galactose- and biotin-derivitized dextran,hydroxypropylmethacrylamide polymers, polyvinylpyrrolidone-polystyrenecopolymers, divinyl ether-maleic acid copolymers, pyran copolymers, orPEG, also have utility as clearing agents in the practice of the presentinvention. In addition, rapidly clearing biotinylated liposomes (highmolecular weight moieties with poor target access) can be derivitizedwith galactose and biotin to produce clearing agents for use in thepractice of the present invention.

A further class of clearing agents useful in the present inventioninvolve small molecules (ranging from about 500 to about 10,000 Dal)derivitized with galactose and biotin that are sufficiently polar to beconfined to the vascular space as an in vivo volume of distribution.More specifically, these agents exhibit a highly charged structure and,as a result, are not readily distributed into the extravascular volume,because they do not readily diffuse across the lipid membranes liningthe vasculature. Exemplary of such clearing agents are mono- orpoly-biotin-derivitized 6,6'- (3,3'-dimethyl1,1'-biphenyl!-4,4'-diyl)bis(azo) bis 4-amino-5-hydroxy-1,3-naphthalenedisulfonic acid! tetrasodium salt, mono- orpoly-biotinyl-galactose-derivitized polysulfated dextran-biotin, mono-or poly-biotinyl-galactose-derivitized dextran-biotin and the like.

The galactose-exposed or -derivitized clearing agents are capable of (1)rapidly and efficiently complexing with the relevant ligand- oranti-ligand-containing conjugates via ligand-anti-ligand affinity; and(2) clearing such complexes from the blood via the galactose receptor, aliver specific degradation system, as opposed to aggregating intocomplexes that are taken up by the generalized RES system, including thelung and spleen. Additionally, the rapid kinetics of galactose-mediatedliver uptake, coupled with the affinity of the ligand-anti-ligandinteraction, allow the use of intermediate or even low molecular weightcarriers.

Non-galactose residue-bearing moieties of low or intermediate molecularweight (ranging from about 40,000 to about 200,000 Dal) localized in theblood may equilibrate with the extravascular space and, therefore, binddirectly to target-associated conjugate, compromising targetlocalization. In addition, aggregation-mediated clearance mechanismsoperating through the RES system are accomplished using a largestoichibmetric excess of clearing agent. In contrast, the rapid bloodclearance of galactose-based clearing agents used in the presentinvention prevents equilibration, and the high affinityligand-anti-ligand binding allows the use of low stoichiometric amountsof such galactose-based clearing agents. This feature further diminishesthe potential for galactose-based clearing agents to compromisetarget-associated conjugate, because the absolute amount of suchclearing agent administered is decreased.

Protein-type and polymer-type non-galactose-based clearing agentsinclude the agents described above, absent galactose exposure orderivitization and the like. These clearing agents act through anaggregation-mediated RES mechanism. In these embodiments of the presentinvention, the clearing agent used will be selected on the basis of thetarget organ to which access of the clearing agent is to be excluded.For example, high molecular weight (ranging from about 200,000 to about1,000,000 Dal) clearing agents will be used when tumor targets or clottargets are involved.

Another class of clearing agents includes agents that do not removecirculating ligand or anti-ligand/targeting moiety-conjugates, butinstead "inactivate" the circulating conjugates by blocking the relevantanti-ligand or ligand binding sites thereon. These "cap-type" clearingagents are preferably small (500 to 10,000 Dal) highly chargedmolecules, which exhibit physical characteristics that dictate a volumeof distribution equal to that of the plasma compartment (i.e., do notextravasate into the extravascular fluid volume). Exemplary cap-typeclearing agents are poly-biotin-derivitized 6,6'- (3,3'-dimethyl1,1'-biphenyl!-4,4'-diyl)bis(azo) bis 4-amino-5-hydroxy-1,3-naphthalenedisulfonic acid! tetrasodium salt, poly-biotinyl-derivitizedpolysulfated dextran-biotin, mono- or poly-biotinyl-derivitizeddextran-biotin and the like.

Cap-type clearing agents are derivitized with the relevant anti-ligandor ligand, and then administered to a recipient of previouslyadministered ligand/or anti-ligand/targeting moiety conjugate. Clearingagent-conjugate binding therefore diminishes the ability of circulatingconjugate to bind any subsequently administered active agent-ligand oractive agent-anti-ligand conjugate. The ablation of active agent bindingcapacity of the circulating conjugate increases the efficiency of activeagent delivery to the target, and increases the ratio of target-boundactive agent to circulating active agent by preventing the coupling oflong-circulating serum protein kinetics with the active agent. Also,confinement of the clearing agent to the plasma compartment preventscompromise of target-associated ligand or anti-ligand.

One embodiment of the present invention in which rapid acting clearingagents are useful is in the delivery of Auger emitters, such as I-125,I-123, Er-165, Sb-119, Hg-197, Ru-97, Tl-201 and I-125 and Br-77, ornucleus-binding drugs to target cell nuclei. In these embodiments of thepresent invention, targeting moieties that localize to internalizingreceptors on target cell surfaces are employed to deliver a targetingmoiety-containing conjugate (i.e., a targeting moiety-anti-ligandconjugate in the preferred two-step protocol) to the target cellpopulation. Such internalizing receptors include EGF receptors,transferrin receptors, HER2 receptors, IL-2 receptors, otherinterleukins and cluster differentiation receptors, somatostatinreceptors, other peptide binding receptors and the like.

After the passage of a time period sufficient to achieve localization ofthe conjugate to target cells, but insufficient to induceinternalization of such targeted conjugates by those cells through areceptor-mediated event, a rapidly acting clearing agent isadministered. In a preferred two-step protocol, an activeagent-containing ligand or anti-ligand conjugate, such as a biotin-Augeremitter or a biotin-nucleus acting drug, is administered as soon as theclearing agent has been given an opportunity to complex with circulatingtargeting moiety-containing conjugate, with the time lag betweenclearing agent and active agent administration being less than about 24hours. In this manner, active agent is readily internalized throughtarget cell receptor-mediated internalization. While circulating Augeremitters are thought to be non-toxic, the rapid, specific targetingafforded by the pretargeting protocols of the present inventionincreases the potential of shorter half-life Auger emitters, such asI-123, which is available and capable of stable binding.

In order to more effectively deliver a therapeutic or diagnostic dose ofradiation to a target site, the radionuclide is preferably retained atthe tumor cell surface. Loss of targeted radiation occurs as aconsequence of metabolic degradation mediated by metabolically activetarget cell types, such as tumor or liver cells.

Preferable agents and protocols within the present invention aretherefore characterized by prolonged residence of radionuclide at thetarget cell site to which the radionuclide has localized and improvedradiation absorbed dose deposition at that target cell site, withdecreased targeted radioactivity loss resulting from metabolism.Radionuclides that are particularly amenable to the practice of thisaspect of the present invention are rhenium, iodine and like "non +3charged" radiometals which exist in chemical forms that easily crosscell membranes and are not, therefore, inherently retained by cells. Incontrast, radionuclides having a +3 charge, such as In-111, Y-90, Lu-177and Ga-67, exhibit natural target cell retention as a result of theircontainment in high charge density chelates.

Evidence exists that streptavidin is resistant to metabolic degradation.Consequently, radionuclides bound directly or indirectly tostreptavidin, rather than, for example, directly to the targetingmoiety, are retained at target cell sites for extended periods of time,as described below in Examples XIV and XV. Streptavidin-associatedradionuclides can be administered in pretargeting protocols or injecteddirectly into lesions.

In addition, streptavidin-associated radionuclides (e.g.,streptavidin-radionuclide and streptavidin-biotin-radionuclide) may beadministered as such (in pretargeting protocols) or as conjugatesincorporating targeting moieties (intralesional injection andpretargeting protocols) specific for stable target cell surface antigens(such as NR-LU-10 antibody, L6, anti-CEA antibodies or the like) ortarget cell internalizing antigens (such as anti-HER2^(neu) ;anti-epidermal growth factor; anti-Lewis Y, including B-1, B-3, BR-64,BR-96 and the like; or the like) to target the streptavidin to theappropriate target cell population.

Streptavidin associated-radionuclides are amenable, for example, tointralesional injection of ovarian cancer lesions studded on theperitoneum and accessible via laparotomy. Another example of anintralesional injection aspect of the present invention involveshepatoma or liver cancer, preferably using a terminalgalactose-streptavidin derivative to bind a radionuclide.

Moreover, high molecular weight carriers, such as biodegradableparticles, dextran, albumin or the like, may be employed (e.g.,conjugated to streptavidin) to limit leakage of the administeredstreptavidin from the injection site. Alternatively, such carriers arebiotinylated, thereby constituting suitable targets or carriers forradionuclide-streptavidin molecules.

The use of streptavidin-associated radionuclides in intralesionalinjection protocols provides the following advantages:

less radionuclide is used to better advantage, because the therapeuticefficacy of the administered radionuclide is improved as a result ofretention at the target cell site;

microdiffusion from the injection site results in expansion of the fieldof radiation deposition;

minimized toxicity and higher dose rate radiation are achieved;

combination with modalities exhibiting disparate toxicity profiles maybe useful;

target sites are imageable post-injection to allow dosimetrydeterminations to be made;

biodegradable (i.e., not requiring removal) retention moiety-carriermolecules can be utilized; and

repeated doses can be injected, because local administration withoutsystemic distribution minimizes antiglobulin response.

The use of streptavidin-associated radionuclides in pretargetingprotocols provides the following advantages:

less radionuclide is used to better advantage, because the therapeuticefficacy of the administered radionuclide is improved as a result ofretention at the target cell site;

target sites are imageable post-injection to allow dosimetrydeterminations to be made;

minimized toxicity and higher dose rate radiation are achieved; and

combination with modalities exhibiting disparate toxicity profiles maybe useful.

In addition, the target cell retention-enhancing aspect of the presentinvention is applicable to a hybrid pretargeting/intralesional injectionprotocol. For example, targeting moiety-biotin conjugate is administeredand an intralesional injection of streptavidin follows after a timesufficient to permit localization of the targeting moiety-biotinconjugate to target cell sites of reasonably determinable location.Next, a radionuclide-biotin molecule is administered, wherein thisadministration is conducted by intralesional, intravenous or otherconvenient route.

Monovalent antibody fragment-streptavidin conjugate may be used topretarget streptavidin, preferably in additional embodiments of thetwo-step aspect of the present invention. Exemplary monovalent antibodyfragments useful in these embodiments are Fv, Fab, Fab' and the like.Monovalent antibody fragments, typically exhibiting a molecular weightranging from about 25 kD (Fv) to about 50 kD (Fab, Fab'), are smallerthan whole antibody and, therefore, are generally capable of greatertarget site penetration. Moreover, monovalent binding can result in lessbinding carrier restriction at the target surface (occurring during useof bivalent antibodies, which bind strongly and adhere to target cellsites thereby creating a barrier to further egress into sublayers oftarget tissue), thereby improving the homogeneity of targeting.

In addition, smaller molecules are more rapidly cleared from arecipient, thereby decreasing the immunogenicity of the administeredsmall molecule conjugate. A lower percentage of the administered dose ofa monovalent fragment conjugate localizes to target in comparison to awhole antibody conjugate. The decreased immunogenicity may permit agreater initial dose of the monovalent fragment conjugate to beadministered, however.

A multivalent, with respect to ligand, moiety is preferably thenadministered. This moiety also has one or more radionuclides associatedtherewith. As a result, the multivalent moiety serves as both a clearingagent for circulating anti-ligand-containing conjugate (throughcross-linking or aggregation of conjugate) and as a therapeutic agentwhen associated with target bound conjugate. In contrast to theinternalization caused by cross-linking described above, cross-linkingat the tumor cell surface stabilizes the monovalent fragment-anti-ligandmolecule and, therefore, enhances target retention, under appropriateconditions of antigen density at the target cell. In addition,monovalent antibody fragments generally do not internalize as dobivalent or whole antibodies. The difficulty in internalizing monovalentantibodies permits cross-linking by a monovalent moiety serves tostabilize the bound monovalent antibody through multipoint binding. Thistwo-step protocol of the present invention has greater flexibility withrespect to dosing, because the decreased fragment immunogenicity allowsmore streptavidin-containing conjugate, for example, to be administered,and the simultaneous clearance and therapeutic delivery removes thenecessity of a separate controlled clearing step.

Another embodiment of the pretargeting methodologies of the presentinvention involves the route of administration of the ligand- oranti-ligand-active agents. In these embodiments of the presentinvention, the active agent-ligand (e.g., radiolabeled biotin) or-anti-ligand is administered intraarterially using an artery supplyingtissue that contains the target. In the radiolabeled biotin example, thehigh extraction efficiency provided by avidin-biotin interactionfacilitates delivery of very high radioactivity levels to the targetcells, provided the radioactivity specific activity levels are high. Thelimit to the amount of radioactivity delivered therefore becomes thebiotin binding capacity at the target (i.e., the amount of antibody atthe target and the avidin equivalent attached thereto).

For these embodiments of the pretargeting methods of the presentinvention, particle emitting therapeutic radionuclides resulting fromtransmutation processes (without non-radioactive carrier forms present)are preferred. Exemplary radionuclides include Y-90, Re-188, At-211,Bi-212 and the like. Other reactor-produced radionuclides are useful inthe practice of these embodiments of the present invention, if they areable to bind in amounts delivering a therapeutically effective amount ofradiation to the target. A therapeutically effective amount of radiationranges from about 1500 to about 10,000 cGy depending upon severalfactors known to nuclear medicine practitioners.

Intraarterial administration pretargeting can be applied to targetspresent in organs or tissues for which supply arteries are accessible.Exemplary applications for intraarterial delivery aspects of thepretargeting methods of the present invention include treatment of livertumors through hepatic artery administration, brain primary tumors andmetastases through carotid artery administration, lung carcinomasthrough bronchial artery administration and kidney carcinomas throughrenal artery administration. Intraarterial administration pretargetingcan be conducted using chemotherapeutic drug, toxin and anti-tumoractive agents as discussed below. High potency drugs, lymphokines, suchas IL-2 and tumor necrosis factor, drug/lymphokine-carrier-biotinmolecules, biotinylated drugs/lymphokines, anddrug/lymphokine/toxin-loaded, biotin-derivitized liposomes are exemplaryof active agents and/or dosage forms useful for the delivery thereof inthe practice of this embodiment of the present invention.

In embodiments of the present invention employing radionuclidetherapeutic agents, the rapid clearance of nontargeted therapeutic agentdecreases the exposure of non-target organs, such as bone marrow, to thetherapeutic agent. Consequently, higher doses of radiation can beadministered absent dose limiting bone marrow toxicity. In addition,pretargeting methods of the present invention optionally includeadministration of short duration bone marrow protecting agents, such asWR 2721. As a result, even higher doses of radiation can be given,absent dose limiting bone marrow toxicity.

While the pretargeting protocols set forth above have been describedprimarily in combination with delivery of a radionuclide diagnostic ortherapeutic moiety, the protocols are amenable to use for delivery ofother moieties, including anti-tumor agents, chemotherapeutic drugs andthe like. For example, most naturally occurring and recombinantcytokines have short in vivo halflives. This characteristic limits theclinical effectiveness of these molecules, because near toxic doses areoften required. Dose-limiting toxicities in humans have been observedupon high dose IL-2 or tumor necrosis factor administrations, forexample.

A protocol, such as administration of streptavidin-targeting moietyconjugate followed by administration of biotinylated cytokine, is alsocontemplated by the present invention. Such pretargeting of anti-ligandserves to improve the performance of cytokine therapeutics by increasingthe amount of cytokine localized to target cells.

Streptavidin-antibody conjugates generally exhibit pharmadokineticssimilar to the native antibody and localize well to target cells,depending upon their construction. Biotinylated cytokines retain a shortin vivo half-life; however, cytokine may be localized to the target as aresult of the affinity of biotin for avidin. In addition, biotin-avidinexperience a pH-dependent dissociation which occurs at a slow rate,thereby permitting a relatively constant, sustained release of cytokineat the target site over time. Also, cytokines complexed to target cellsthrough biotin-avidin association are available for extraction andinternalization by cells involved in cellular-mediated cytotoxicity.

A pre-formed antibody-streptavidin-biotin-cytokine preparation may alsobe employed in the practice of these methods of the present invention.In addition, a three-step protocol of the present invention may also beemployed to deliver a cytokine, such as IL-2, to a target site.

Other anti-tumor agents that may be delivered in accordance with thepretargeting techniques of the present invention are selecting,including L-selectin, P-selectin and E-selectin. The presence ofcytokines stimulates cells, such as endothelial cells, to expressselecting on the surfaces thereof. Selectins bind to white blood cellsand aid in delivering white blood cells where they are needed.Consequently, a protocol, such as administration of streptavidin- oravidin-targeting moiety conjugate followed by administration ofbiotinylated selecting, is also contemplated by the present invention.Such pretargeting of anti-ligand serves to improve the performance ofselectin therapeutics by increasing the amount of selectin localized totarget cells. In this manner, the necessity of cytokine induction ofselectin expression is obviated by the localization and retention ofselectin at a target cell population.

Chemotherapeutic drugs also generally exhibit short in vivo half-livesat a therapeutically effective dose. Consequently, another example of aprotocol of the present invention includes administration ofavidin-targeting moiety conjugate followed by administration of abiotin-chemotherapeutic drug conjugate or complex, such as adrug-carrier-biotin complex. A three-step protocol of the presentinvention may also be employed to deliver a chemotherapeutic drug, suchas methotrexate, adriamycin, high potency adriamycin analogs,trichothecenes, potent enediynes, such as esperamycins andcalicheamycins, cytoxan, vinca alkaloids, actinamycin D, taxol, taxotereor the like to a target site.

The invention is further described through presentation of the followingexamples. These examples are offered by way of illustration, and not byway of limitation.

EXAMPLE I Synthesis of a Chelate-Biotin Conjugate

A chelating compound that contains an N₃ S chelating core was attachedvia an amide linkage to biotin. Radiometal labeling of an exemplarychelate-biotin conjugate is illustrated below. ##STR2##

The spacer group "X" permits the biotin portion of the conjugate to besterically available for avidin binding. When "R¹ " is a carboxylic acidsubstituent (for instance, CH₂ COOH), the conjugate exhibits improvedwater solubility, and further directs in vivo excretion of theradiolabeled biotin conjugate toward renal rather than hepatobiliaryclearance.

Briefly, N-α-Cbz-N-Σ-t-BOC protected lysine was converted to thesuccinimidyl ester with NHS and DCC, and then condensed with asparticacid β-t-butyl ester. The resultant dipeptide was activated with NHS andDCC, and then condensed with glycine t-butyl ester. The Cbz group wasremoved by hydrogenolysis, and the amine was acylated usingtetrahydropyranyl mercaptoacetic acid succinimidyl ester, yieldingS-(tetrahydropyranyl)-mercaptoacetyl-lysine. Trifluoroacetic acidcleavage of the N-T-BOC group and t-butyl esters, followed bycondensation with LC-biotin-NHS ester provided (Σ-caproylamidebiotin)-aspartyl glycine. This synthetic method is illustrated below.##STR3##

¹ H NMR: (CD₃ OD, 200 MHz Varian): 1.25-1.95 (m; 24H), 2.15-2.25 (broadt, 4H), 2.65-3.05 (m, 4H), 3.30-3.45 (dd, 2H), 3.50-3.65 (ddd, 2H), 3.95(broad s, 2H), 4.00-4.15 (m, 1H), 4.25-4.35 (m; 1H), 4.45-4.55 (m, 1H),4.7-5.05 (m overlapping with HOD).

Elemental Analysis: C, H, N for C₃₅ H₅₇ N₇ O₁₁ S₂.H₂ O calculated:50.41, 7.13, 11.76 found: 50.13, 7.14, 11.40

EXAMPLE II Preparation of a Technetium or Rhenium RadolabeledChelate-Biotin Conjugate

The chelate-biotin conjugate of Example I was radiolabeled with either^(99m) Tc pertechnetate or ¹⁸⁶ Re perrhenate. Briefly, ^(99m) Tcpertechnetate was reduced with stannous chloride in the presence ofsodium gluconate to form an intermediate Tc-gluconate complex. Thechelate-biotin conjugate of Example I was added and heated to 100° C.for 10 min at a pH of about 1.8 to about 3.3. The solution wasneutralized to a pH of about 6 to about 8, and yielded an N₃S-coordinated ^(99m) Tc-chelate-biotin conjugate. C-18 HPLC gradientelution using 5-60% acetonitrile in 1% acetic acid demonstrated twoanomers at 97% or greater radiochemical yield using δ (gamma ray)detection.

Alternatively, ¹⁸⁶ Re perrhenate was spiked with cold ammoniumperrhenate, reduced with stannous chloride, and complexed with citrate.The chelate-biotin conjugate of Example I was added and heated to 90° C.for 30 min at a pH of about 2 to 3. The solution was neutralized to a pHof about 6 to about 8, and yielded an N₃ S-coordinated ¹⁸⁶Re-chelate-biotin conjugate. C-18 HPLC gradient elution using 5-60%acetonitrile in 1% acetic acid resulted in radiochemical yields of85-90%. Subsequent purification over a C-18 reverse phase hydrophobiccolumn yielded material of 99% purity.

EXAMPLE III In Vitro Analysis of Radiolabeled Chelate-Biotin Conjugates

Both the ^(99m) Tc- and ¹⁸⁶ Re-chelate-biotin conjugates were evaluatedin vitro. When combined with excess avidin (about 100-fold molarexcess), 100% of both radiolabeled biotin conjugates complexed withavidin.

A ^(99m) Tc-biotin conjugate was subjected to various chemical challengeconditions. Briefly, ^(99m) Tc-chelate-biotin conjugates were combinedwith avidin and passed over a 5 cm size exclusion gel filtration column.The radiolabeled biotin-avidin complexes were subjected to variouschemical challenges (see Table 1), and the incubation mixtures werecentrifuged through a size exclusion filter. The percent ofradioactivity retained (indicating avidin-biotin-associated radiolabel)is presented in Table 1. Thus, upon chemical challenge, the radiometalremained associated with the macromolecular complex.

                  TABLE 1    ______________________________________    Chemical Challenge of .sup.99m Tc-Chelate-    Biotin-Avidin Complexes    Challenge             % Radioactivity Retained    Medium      pH        1 h, 37° C.                                    18 h, RT    ______________________________________    PBS         7.2       99        99    Phosphate   8.0       97        97    10 mM cysteine                8.0       92        95    10 mM DTPA  8.0       99        98    0.2M carbonate                10.0      97        94    ______________________________________

In addition, each radiolabeled biotin conjugate was incubated at about50 μg/ml with serum; upon completion of the incubation, the samples weresubjected to instant thin layer chromatography (ITLC) in 80% methanol.Only 2-4% of the radioactivity remained at the origin (i.e., associatedwith protein); this percentage was unaffected by the addition ofexogenous biotin. When the samples were analyzed using size exclusionH-12 PPLC with 0.2 M phosphate as mobile phase, no association ofradioactivity with serum macromolecules was observed.

Each radiolabeled biotin conjugate was further examined using acompetitive biotin binding assay. Briefly, solutions containing varyingratios of D-biotin to radiolabeled biotin conjugate were combined withlimiting avidin at a constant total biotin:avidin ratio. Avidin bindingof each radiolabeled biotin conjugate was determined by ITLC, and wascompared to the theoretical maximum stoichiometric binding (asdetermined by the HABA spectrophotometric assay of Green, Biochem. J.94:23c-24c, 1965). No significant difference in avidin binding wasobserved between each studied in an animal model of a three-stepantibody radiolabeled biotin conjugate and D-biotin.

EXAMPLE IV In Vivo Analysis of Radiolabeled Chelate-Biotin ConjugatesAdministered After Antibody Pretargeting

The ¹⁸⁶ Re-chelate-biotin conjugate of Example I was studied in ananimal model of a three-step antibody pretargeting protocol. Generally,this protocol monoclonal antibody; (ii) administration of avidin forformation of a "sandwich" at the target site and for clearance ofresidual circulating biotinylated conjugate for target site localizationand rapid blood clearance.

A. Preparation and Characterization of Biotinylated Antibody

Biotinylated NR-LU-10 was prepared according to either of the followingprocedures. The first procedure involved derivitization of antibody vialysine ε-amino groups. NR-LU-10 was radioiodinated at tyrosines usingchloramine T and either ¹²⁵ I or ¹³¹ I sodium iodide. The radioiodinatedantibody (5-10 mg/ml) was then biotinylated using biotinamido caproateNHS ester in carbonate buffer, pH 8.5, containing 5% DMSO, according tothe scheme below. ##STR4##

The impact of lysine biotinylation on antibody immunoreactivity wasexamined. As the molar offering of biotinantibody increased from 5:1 to40:1, biotin incorporation increased as expected (measured using theHABA assay and pronase-digested product) (Table 2, below). Percent ofbiotinylated antibody immunoreactivity as compared to native antibodywas assessed in a limiting antigen ELISA assay. The immunoreactivitypercentage dropped below 70% at a measured derivitization of 11.1:1;however, at this level of derivitization, no decrease was observed inantigen-positive cell binding (performed with LS-180 tumor cells atantigen excess). Subsequent experiments used antibody derivitized at abiotinoantibody ratio of 10:1.

                  TABLE 2    ______________________________________    Effect of Lysine Biotinylation    on Immunoreactivity    Molar     Measured    Offering  Derivitization                            Immunoassessment (%)    (Biotins/Ab)              (Biotins/Ab)  ELISA   Cell Binding    ______________________________________     5:1      3.4           86    10:1      8.5           73      100    13:1      11.1          69      102    20:1      13.4          36      106    40:1      23.1          27    ______________________________________

Alternatively, NR-LU-10 was biotinylated using thiol groups generated byreduction of cystines. Derivitization of thiol groups was hypothesizedto be less compromising to antibody immunoreactivity. NR-LU-10 wasradioiodinated using p-aryltin phenylate NHS ester (PIP-NHS) and either¹²⁵ I or ¹³¹ I sodium iodide. Radioiodinated NR-LU-10 was incubated with25 mM dithiothreitol and purified using size exclusion chromatography.The reduced antibody (containing free thiol groups) was then reactedwith a 10- to 100-fold molar excess of N-iodoacetyl-n'-biotinyl hexylenediamine in phosphate-buffered saline (PBS), pH 7.5, containing 5% DMSO(v/v).

                  TABLE 3    ______________________________________    Effect of Thiol Biotinylation    on Immunoreactivity    Molar     Measured    Offering  Derivitization                            Immunoassessment (%)    (Biotins/Ab)              (Biotins/Ab)  ELISA   Cell Binding    ______________________________________     10:1     4.7           114     50:1     6.5           102     100    100:1     6.1            95     100    ______________________________________

As shown in Table 3, at a 50:1 or greater biotin:antibody molaroffering, only 6 biotins per antibody were incorporated. No significantimpact on immunoreactivity was observed.

The lysine- and thiol-derivitized biotinylated antibodies ("antibody(lysine)" and "antibody (thiol)", respectively) were compared. Molecularsizing on size exclusion FPLC demonstrated that both biotinylationprotocols yielded monomolecular (monomeric) IgGs. Biotinylated antibody(lysine) had an apparent molecular weight of 160 kD, while biotinylatedantibody (thiol) had an apparent molecular weight of 180 kD. Reductionof endogenous sulfhydryls to thiol groups, followed by conjugation withbiotin, may produce a somewhat unfolded macromolecule. If so, theantibody (thiol) may display a larger hydrodynamic radius and exhibit anapparent increase in molecular weight by chromatographic analysis. Bothbiotinylated antibody species exhibited 98% specific binding toimmobilized avidin-agarose.

Further comparison of the biotinylated antibody species was performedusing non-reducing SDS-PAGE, using a 4% stacking gel and a 5% resolvinggel. Biotinylated samples were either radiolabeled or unlabeled and werecombined with either radiolabeled or unlabeled avidin or streptavidin.Samples were not boiled prior to SDS-PAGE analysis. The native antibodyand biotinylated antibody (lysine) showed similar migrations; thebiotinylated antibody (thiol) produced two species in the 50-75 kDrange. These species may represent two thiol-capped species. Under theseSDS-PAGE conditions, radiolabeled streptavidin migrates as a 60 kDtetramer. When 400 μg/ml radiolabeled streptavidin was combined with 50μg/ml biotinylated antibody (analogous to "sandwiching" conditions invivo), both antibody species formed large molecular weight complexes.However, only the biotinylated antibody (thiol)-streptavidin complexmoved from the stacking gel into the resolving gel, indicating adecreased molecular weight as compared to the biotinylated antibody(lysine)-streptavidin complex.

B. Blood Clearance of Biotinylated Antibody Species

Radioiodinated biotinylated NR-LU-10 (lysine or thiol) was intravenouslyadministered to non-tumored nude mice at a dose of 100 μg. At 24 hpost-administration of radioiodinated biotinylated NR-LU-10, mice wereintravenously injected with either saline or 400 μg of avidin. Withsaline administration, blood clearances for both biotinylated antibodyspecies were biphasic and similar to the clearance of native NR-LU-10antibody.

In the animals that received avidin intravenously at 24 h, thebiotinylated antibody (lysine) was cleared (to a level of 5% of injecteddose) within 15min of avidin administration (avidin:biotin=10:1). Withthe biotinylated antibody (thiol), avidin administration (10:1 or 25:1)reduced the circulating antibody level to about 35% of injected doseafter two hours. Residual radiolabeled antibody activity in thecirculation after avidin administration was examined in vitro usingimmobilized biotin. This analysis revealed that 85% of the biotinylatedantibody was complex ed with avidin. These data suggest that thebiotinylated antibody (thiol)-avidin complexes that were formed wereinsufficiently crosslinked to be cleared by the RES.

Blood clearance and biodistribution studies of biotinylated antibody(lysine) 2 h post-avidin or post-saline administration were performed.Avidin administration significantly reduced the level of biotinylatedantibody in the blood (see FIG. 1); and increased the level ofbiotinylated antibody in the liver and spleen. Kidney levels ofbiotinylated antibody were similar.

EXAMPLE V In Vivo Characterization of ¹⁸⁶ Re-Chelate-Biotin ConjugatesIn a Three-Step Pretargeting Protocol

A ¹⁸⁶ Re-chelate-biotin conjugate of Example I (MW≈1000; specificactivity=1-2 mCi/mg) was examined in a three-step pretargeting protocolin an animal model. More specifically, 18-22 g female nude mice wereimplanted subcutaneously with LS-180 human colon tumor xenografts,yielding 100-200 mg tumors within 10 days of implantation.

NR-LU-10 antibody (MW≈150 kD) was radiolabeled with ¹²⁵ I/Chloramine Tand -biotinylated via lysine residues (as described in Example IV.A,above). Avidin (MW≈66 kD) was radiolabeled with ¹³¹ I/PIP-NHS (asdescribed for radioiodination of NR-LU-10 in Example IV.A., above). Theexperimental protocol was as follows:

    ______________________________________    Group 1:    Time 0, inject 100 μg .sup.125 I-labeled,                biotinylated NR-LU-10                Time 24 h, inject 400 μg .sup.131 I-labeled                avidin                Time 26 h, inject 60 μg .sup.186 Re-chelate-                biotin conjugate    Group 2:    Time 0, inject 400 μg .sup.131 I-labeled avidin    (control)   Time 2 h, inject 60 μg .sup.186 Re-chelate-                biotin conjugate    Group 3:    Time 0, inject 60 μg .sup.186 Re-chelate-    (control)   biotin conjugate    ______________________________________

The three radiolabels employed in this protocol are capable of detectionin the presence of each other. It is also noteworthy that the sizes ofthe three elements involved are logarithmicallydifferent--antibody≈150,000; avidin≈66,000, and biotin≈1,000.Biodistribution analyses were performed at 2, 6, 24, 72 and 120 h afteradministration of the ¹⁸⁶ -Re-chelate-biotin conjugate.

Certain preliminary studies were performed in the animal model prior toanalyzing the ¹⁸⁶ Re-chelate-biotin conjugate in a three-steppretargeting protocol. First, the effect of biotinylated antibody onblood clearance of avidin was examined. These experiments showed thatthe rate and extent of avidin clearance was similar in the presence orabsence of biotinylated antibody. Second, the effect of biotinylatedantibody and avidin on blood clearance of the ¹⁸⁶ Re-chelate-biotinconjugate was examined; blood clearance was similar in the presence orabsence of biotinylated antibody and avidin. Further, antibodyimmunoreactivity was found to be uncompromised by biotinylation at thelevel tested.

Third, tumor uptake of biotinylated antibody administered at time 0 orof avidin administered at time 24 h was examined. The results of thisexperimentation are shown in FIG. 1. At 25 h, about 350 pmol/gbiotinylated antibody was present at the tumor; at 32 h the level wasabout 300 pmol/g; at 48 h, about 200 pmol/g; and at 120 h, about 100pmol/g. Avidin uptake at the same time points was about 250, 150, 50 and0 pmol/g, respectively. From the same experiment, tumor to blood ratioswere determined for biotinylated antibody and for avidin. From 32 h to120 h, the ratios of tumor to blood were very similar.

Rapid and efficient removal of biotinylated antibody from the blood bycomplexation with avidin was observed. Within two hours of avidinadministration, a 10-fold reduction in blood pool antibody concentrationwas noted (FIG. 1), resulting in a sharp increase in tumor to bloodratios. Avidin is cleared rapidly, with greater than 90% of the injecteddose cleared from the blood within 1 hour after administration. TheRe-186-biotin chelate is also very rapidly cleared, with greater than99% of the injected dose cleared from the blood by 1 hour afteradministration.

The three-step pretargeting protocol (described for Group 1, above) wasthen examined. More specifically, tumor uptake of the ¹⁸⁶Re-chelate-biotin conjugate in the presence or absence of biotinylatedantibody and avidin was determined. In the absence of biotinylatedantibody and avidin, the ¹⁸⁶ Re-chelate-biotin conjugate displayed aslight peak 2 h post-injection, which was substantially cleared from thetumor by about 5 h. In contrast, at 2 h post-injection in the presenceof biotinylated antibody and avidin (specific), the ¹⁸⁶Re-chelate-biotin conjugate reached a peak in tumor approximately 7times greater than that observed in the absence of biotinylated antibodyand avidin. Further, the specifically bound ¹⁸⁶ Re-chelate-biotinconjugate was retained at the tumor at significant levels for more than50 h. Tumor to blood ratios determined in the same experiment increasedsignificantly over time (i.e., T:B=≈8 at 30 h; ≈15 at 100 h; ≈35 at 140h).

Tumor uptake of the ¹⁸⁶ Re-chelate-biotin conjugate has further beenshown to be dependent on the dose of biotinylated antibody administered.At 0 μg of biotinylated antibody, about 200 pmol/g of ¹⁸⁶Re-chelate-biotin conjugate was present at the tumor at 2 h afteradministration; at 50 μg antibody, about 500 pmol/g of ¹⁸⁶Re-chelate-biotin conjugate; and at 100 μg antibody, about 1,300 pmol/gof ¹⁸⁶ Re-chelate-biotin conjugate.

Rhenium tumor uptake via the three-step pretargeting protocol wascompared to tumor uptake of the same antibody radiolabeled throughchelate covalently attached to the antibody (conventional procedure).The results of this comparison are depicted in FIG. 2. Blood clearanceand tumor uptake were compared for the chelate directly labeled rheniumantibody conjugate and for the three-step pretargeted sandwich. Areasunder the curves (AUC) and the ratio of AUC_(tumor) /AUC_(blood) weredetermined. For the chelate directly labeled rhenium antibody conjugate,the ratio of AUC_(tumor) /AUC_(blood) =24055/10235 or 2.35; for thethree-step pretargeted sandwich, the ratio of AUC_(tumor) /AUC_(blood)=46764/6555 or 7.13.

Tumor uptake results are best taken in context with radioactivityexposure to the blood compartment, which directly correlates with bonemarrow exposure. Despite the fact that 100-fold more rhenium wasadministered to animals in the three-step protocol, the very rapidclearance of the small molecule (Re-186-biotin) from the blood minimizesthe exposure to Re-186 given in this manner. In the same matchedantibody dose format, direct labeled (conventional procedure) NR-LU-10whole antibody yielded greater exposure to rhenium than did the 100-foldhigher dose given in the three-step protocol. A clear increase in thetargeting ratio (tumor exposure to radioactivity:blood exposure toradioactivity--AUC_(tumor) :AUC_(blood)) was observed for three-steppretargeting (approximately 7:1) in comparison to the direct labeledantibody approach (approximately 2.4:1).

EXAMPLE VI Preparation of Chelate-Biotin Conjugates Having ImprovedBiodistribution Properties

The biodistribution of ¹¹¹ In-labeled-biotin derivatives varies greatlywith structural changes in the chelate and the conjugating group.Similar structural changes may affect the biodistribution of technetium-and rhenium-biotin conjugates. Accordingly, methods for preparingtechnetium- and rhenium-biotin conjugates having optimal clearance fromnormal tissue are advantageous.

A. Neutral MAMA Chelate/Conjugate

A neutral MAMA chelate-biotin conjugate is prepared according to thefollowing scheme. ##STR5## The resultant chelate-biotin conjugate showssuperior kidney excretion. Although the net overall charge of theconjugate is neutral, the polycarboxylate nature of the moleculegenerates regions of hydrophilicity and hydrophobicity. By altering thenumber and nature of the carboxylate groups within the conjugate,excretion may be shifted from kidney to gastrointestinal routes. Forinstance, neutral compounds are generally cleared by the kidneys;anionic compounds are generally cleared through the GI system.

B. Polylysine Derivitization

Conjugates containing polylysine may also exhibit beneficialbiodistribution properties. With whole antibodies, derivitization withpolylysine may skew the biodistribution of conjugate toward liveruptake. In contrast, derivitization of Fab fragments with polylysineresults in lower levels of both liver and kidney uptake; blood clearanceof these conjugates is similar to that of Fab covalently linked tochelate. An exemplary polylysine derivitized chelate-biotin conjugate isillustrated below. ##STR6## Inclusion of polylysine inradiometal-chelate-biotin conjugates is therefore useful for minimizingor eliminating RES sequestration while maintaining good liver and kidneyclearance of the conjugate. For improved renal excretion properties,polylysine derivatives are preferably succinylated followingbiotinylation. Polylysine derivatives offer the further advantages of:(1) increasing the specific activity of the radiometal-chelate-biotinconjugate; (2) permitting control of rate and route of blood clearanceby varying the molecular weight of the polylysine polymer; and (3)increasing the circulation half-life of the conjugate for optimal tumorinteraction.

Polylysine derivitization is accomplished by standard methodologies.Briefly, poly-L-lysine is acylated according to standard amino groupacylation procedures (aqueous bicarbonate buffer, pH 8, added biotin-NHSester, followed by chelate NHS ester). Alternative methodology involvesanhydrous conditions using nitrophenyl esters in DMSO and triethylamine. The resultant conjugates are characterized by UV and NMR spectra.

The number of biotins attached to polylysine is determined by the HABAassay. Spectrophotometric titration is used to assess the extent ofamino group derivitization. The radiometal-chelate-biotin conjugate ischaracterized by size exclusion.

C. Cleavable Linkage

Through insertion of a cleavable linker between the chelate and biotinportion of a radiometal-chelate-biotin conjugate, retention of theconjugate at the tumor relative to normal tissue may be enhanced. Morespecifically, linkers that are cleaved by enzymes present in normaltissue but deficient or absent in tumor tissue can increase tumorretention. As an example, the kidney has high levels of γ-glutamyltransferase; other normal tissues exhibit in vivo cleavage of γ-glutamylprodrugs. In contrast, tumors are generally deficient in enzymepeptidases. The glutamyl-linked biotin conjugate depicted below iscleaved in normal tissue and retained in the tumor. ##STR7##

D. Serine Linker With O-Polar Substituent

Sugar substitution of N₃ S chelates renders such chelates water soluble.Sulfonates, which are fully ionized at physiological pH, improve watersolubility of the chelate-biotin conjugate depicted below. ##STR8## R=asugar as ribose or glucose or SO₂ OH X=(CH₂)₀ or CO(CH:₂)₄

This compound is synthesized according to the standard reactionprocedures. Briefly, biocytin is condensed with N-t-BOC-(O-sulfonate orO-glucose) serine NHS ester to give N-t-BOC-(O-sulfonate or O-glucose)serine biocytinamide. Subsequent cleavage of the N-t-BOC group with TFAand condensation with ligand NHS ester in DMF with triethylamineprovides ligand-amidoserine(O-sulfonate or O-glucose)biocytinamide.

EXAMPLE VII Preparation and Characterization of PIP-RadioiodinatedBiotin

Radioiodinated biotin derivatives prepared by exposure of poly-L-lysineto excess NHS-LC-biotin and then to Bolton-Hunter N-hydroxysuccinimideesters in DMSO has been reported. After purification, this product wasradiolabeled by the iodogen method (see, for instance, Del Rosario etal., J. Nucl. Med. 32:5, 1991, 993 (abstr.)). Because of the highmolecular weight of the resultant radioiodinated biotin derivative, onlylimited characterization of product (i.e., radio-HPLC and binding toimmobilized streptavidin) was possible.

Preparation of radioiodinated biotin according to the present inventionprovides certain advantages. First, the radioiodobiotin derivative is alow molecular weight compound that is amenable to complete chemicalcharacterization. Second, the disclosed methods for preparation involvea single step and eliminate the need for a purification step.

Briefly, iodobenzamide derivatives corresponding to biocytin (R=COOH)and biotinamidopentylamine (R=H) were prepared according to thefollowing scheme. In this scheme, "X" may be any radiohalogen, including¹²⁵ I, ¹³¹ I, ¹²³ I, ²¹¹ At and the like. ##STR9## Preparation of 1 wasgenerally according to Wilbur et al., J. Nucl. Med. 30:0216-26, 1989,using a tributyltin intermediate. Water soluble carbodiimide was used inthe above-depicted reaction, since the NHS ester 1 formed intractablemixtures with DCU. The NHS ester was not compatible with chromatography;it was insoluble in organic and aqueous solvents and did not react withbiocytin in DMF or in buffered aqueous acetonitrile. The reactionbetween 1 and biocytin or 5-(biotinamido) pentylamine was sensitive tobase. When the reaction of 1 and biocytin or the pentylamine wasperformed in the presence of triethylamine in hot DMSO, formation ofmore than one biotinylated product resulted. In contrast, the reactionwas extremely clean and complete when a suspension of 1 and biocytin (4mg/ml) or the pentylamine (4 mg/ml) was heated in DHSO at 117° C. forabout 5 to about 10 min. The resultant ¹²⁵ I-biotin derivatives wereobtained in 94% radiochemical yield. Optionally, the radioiodinatedproducts may be purified using C-18 HPLC and a reverse phase hydrophobiccolumn. Hereinafter, the resultant radioiodinated products 2 arereferred to as PIP-biocytin (R=COOH) and PIP-pentylamine (R=H).

Both iodobiotin derivatives 2 exhibited ≧95% binding to immobilizedavidin. Incubation of the products 2 with mouse serum resulted in noloss of the ability of 2 to bind to immobilized avidin. Biodistributionstudies of 2 in male BALB/c mice showed rapid clearance from the blood(similar to ¹⁸⁶ Re-chelate-biotin conjugates described above). Theradioiodobiotin 2 had decreased hepatobiliary excretion as compared tothe ¹⁸⁶ Re-chelate-biotin conjugate, urinary excretion was increased ascompared to the ¹⁸⁶ Re-chelate-biotin conjugate. Analysis of urinarymetabolites of 2 indicated deiodination and cleavage of the biotin amidebond; the metabolites showed no binding to immobilized avidin. Incontrast, metabolites of the ¹⁸⁶ Re-chelate-biotin conjugate appear tobe excreted in urine as intact biotin conjugates. Intestinal uptake of 2is <50% that of the ¹⁸⁶ Re-chelate-biotin conjugate. Thesebiodistribution properties of 2 provided enhanced whole body clearanceof radioisotope and indicate the advantageous use of 2 withinpretargeting protocols.

¹³¹ I-PIP-biocytin was evaluated in a two-step pretargeting procedure intumor-bearing mice. Briefly, female nude mice were injectedsubcutaneously with LS-180 tumor cells; after 7 d, the mice displayed50-100 mg tumor xenografts. At t=0, the mice were injected with 200 μgof NR-LU-10-avidin conjugate labeled with ¹²⁵ I using PIP-NHS (seeExample IV.A.). At t=36 h, the mice received 42 μg of ¹³¹I-PIP-biocytin. The data showed immediate, specific tumor localization,corresponding to ≈1.5 ¹³¹ I-PIP-biocytin molecules per avidin molecule.

The described radiohalogenated biotin compounds are amenable to the sametypes of modifications described in Example VI above for ¹⁸⁶Re-chelate-biotin conjugates. In particular, the followingPIP-polylysine-biotin molecule is made by trace labeling polylysine with¹²⁵ I-PIP, followed by extensive biotinylation of the polylysine.##STR10## Assessment ¹²⁵ I binding to immobilized avidin ensures thatall radioiodinated species also contain at least an equivalent ofbiotin.

EXAMPLE VIII Preparation of Biotinylated Antibody (Thiol) ThroughEndogenous Antibody Sulfhydryl Groups Or Sulfhydryl-Generating Compounds

Certain antibodies have available for reaction endogenous sulfhydrylgroups. If the antibody to be biotinylated contains endogenoussulfhydryl groups, such antibody is reacted withN-iodoacetyl-n'-biotinyl hexylene diamine (as described in ExampleIV.A., above). The availability of one or more endogenous sulfhydrylgroups obviates the need to expose the antibody to a reducing agent,such as DTT, which can have other detrimental effects on thebiotinylated antibody.

Alternatively, one or more sulfhydryl groups are attached to a targetingmoiety through the use of chemical compounds or linkers that contain aterminal sulfhydryl group. An exemplary compound for this purpose isiminothiolane. As with endogenous sulfhydryl groups (discussed above),the detrimental effects of reducing agents on antibody are therebyavoided.

EXAMPLE IX Two-Step Pretargeting Methodology That Does Not InduceInternalization

A NR-LU-13-avidin conjugate is prepared as follows. Initially, avidin isderivitized with N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). SMCC-derivedavidin is then incubated with NR-LU-13 in a 1:1 molar ratio at pH 8.5for 16 h. Unreacted NR-LU-13 and SMCC-derived avidin are removed fromthe mixture using preparative size exclusion HPLC. Two conjugates areobtained as products--the desired 1:1 NR-LU-13-avidin conjugate as themajor product; and an incompletely characterized component as the minorproduct.

A ^(99m) Tc-chelate-biotin conjugate is prepared as in Example II,above. The NR-LU-13-avidin conjugate is administered to a recipient andallowed to clear from the circulation. One of ordinary skill in the artof radioimmunoscintigraphy is readily able to determine the optimal timefor NR-LU-13-avidin conjugate tumor localization and clearance from thecirculation. At such time, the ^(99m) Tc-chelate-biotin conjugate isadministered to the recipient. Because the ^(99m) Tc-chelate-biotinconjugate has a molecular weight of ≈1,000, crosslinking ofNR-LU-13-avidin molecules on the surface of the tumor cells isdramatically reduced or eliminated. As a result, the ^(99m) Tcdiagnostic agent is retained at the tumor cell surface for an extendedperiod of time. Accordingly, detection of the diagnostic agent byimaging techniques is optimized; further, a lower dose of radioisotopeprovides an image comparable to that resulting from the typicalthree-step pretargeting protocol.

Optionally, clearance of NR-LU-13-avidin from the circulation may beaccelerated by plasmapheresis in combination with a biotin affinitycolumn. Through use of such column, circulating NR-LU-13-avidin will beretained extracorporeally, and the recipient's immune system exposure toa large, proteinaceous immunogen (i.e., avidin) is minimized.

Clearance of NR-LU-13-avidin may also be accelerated by an arteriallyinserted proteinaceous or polymeric multiloop device. A catheter-likedevice, consisting of thin loops of synthetic polymer or protein fibersderivitized with biotin, is inserted into a major artery (e.g., femoralartery) to capture NR-LU-13-avidin. Since the total blood volume passesthrough a major artery every 70 seconds, the in situ clearing device iseffective to reduce circulating NR-LU-13-avidin within a short period oftime. This device offers the advantages that NR-LU-13-avidin is notprocessed through the RES; removal of NR-LU-13-avidin is controllableand measurable; and fresh devices with undiminished binding capacity areinsertable as necessary. This methodology is also useful withintraarterial administration embodiments of the present invention.

An alternative procedure for clearing NR-LU-13-avidin from thecirculation without induction of internalization involves administrationof biotinylated, high molecular weight molecules, such as liposomes, IgMand other molecules that are size excluded from ready permeability totumor sites. When such biotinylated, high molecular weight moleculesaggregate with NR-LU-13-avidin, the aggregated complexes are readilycleared from the circulation via the RES.

EXAMPLE X Enhancement of Therapeutic Agent Internalization ThroughAvidin Crosslinking

The ability of multivalent avidin to crosslink two or more biotinmolecules (or chelate-biotin conjugates) is advantageously used toimprove delivery of therapeutic agents. More specifically, avidincrosslinking induces internalization of crosslinked complexes at thetarget cell surface.

Biotinylated NR-CO-04 (lysine) is prepared according to the methodsdescribed in Example IV.A., above. Doxorubicin-avidin conjugates areprepared by standard conjugation chemistry. The biotinylated NR-CO-04 isadministered to a recipient and allowed to clear from the circulation.One of ordinary skill in the art of radioimmunotherapy is readily ableto determine the optimal time for biotinylated NR-CO-04 tumorlocalization and clearance from the circulation. At such time, thedoxorubicin-avidin conjugate is administered to the recipient. Theavidin portion of the doxorubicin-avidin conjugate crosslinks thebiotinylated NR-CO-04 on the cell surface, inducing internalization ofthe complex. Thus, doxorubicin is more efficiently delivered to thetarget cell.

In a first alternative protocol, a standard three-step pretargetingmethodology is used to enhance intracellular delivery of a drug to atumor target cell. By analogy to the description above, biotinylatedNR-LU-05 is administered, followed by avidin (for blood clearance and toform the middle layer of the sandwich at the target cell-boundbiotinylated antibody). Shortly thereafter, and prior to internalizationof the biotinylated NR-LU-05-avidin complex; a methotrexate-biotinconjugate is administered.

In a second alternative protocol, biotinylated NR-LU-05 is furthercovalently linked to methotrexate. Subsequent administration of avidininduces internalization of the complex and enhances intracellulardelivery of drug to the tumor target cell.

In a third alternative protocol, NR-CO-04-avidin is administered-to arecipient and allowed to clear from the circulation and localize at thetarget site. Thereafter, a polybiotinylated species (such asbiotinylated poly-L-lysine, as in Example IV.B., above) is administered.In this protocol, the drug to be delivered may be covalently attached toeither the antibody-avidin component or to the polybiotinylated species.The polybiotinylated species induces internalization of the(drug)-antibody-avidin-polybiotin-(drug) complex.

EXAMPLE XI Targeting Moiety-Anti-Ligand Conjugate for Two-StepPretargeting In Vivo

A. Preparation of SMCC-derivitized streptavidin.

31 mg (0.48 μmol) streptavidin was dissolved in 9.0 ml PBS to prepare afinal solution at 3.5 mg/ml. The pH of the solution was adjusted to 8.5by addition of 0.9 ml of 0.5 M borate buffer, pH 8.5. A DMSO solution ofSMCC (3.5 mg/ml) was prepared, and 477 μl (4.8 μmol) of this solutionwas added dropwise to the vortexing protein solution. After 30 minutesof stirring, the solution was purified by G-25 (PD-10, Pharmacia,Piscataway, N.J.) column chromatography to remove unreacted orhydrolyzed SMCC. The purified SMCC-derivitized streptavidin was isolated(28 mg, 1.67 mg/ml).

B. Preparation of DTT-reduced NR-LU-10. To 77 mg NR-LU-10 (0.42 μmol) in15.0 ml PBS was added 1.5 ml of 0.5 M borate buffer, pH 8.5. A DTTsolution, at 400 mg/ml (165 μl) was added to the protein solution. Afterstirring at room temperature for 30 minutes, the reduced antibody waspurified by G-25 size exclusion chromatography. Purified DTT-reducedNR-LU-10 was obtained (74 mg, 2.17 mg/ml).

C. Conjugation of SMCC-streptavidin to DTT-reduced NR-LU-10. DTT-reducedNR-LU-10 (63 mg, 29 ml, 0.42 μmol) was diluted with 44.5 ml PBS. Thesolution of SMCC-streptavidin (28 mg, 17 ml, 0.42 μmol) was addedrapidly to the stirring solution of NR-LU-10. Total proteinconcentration in the reaction mixture was 1.0 mg/ml. The progress of thereaction was monitored by HPLC (Zorbax® GF-250, available from MacMod).After approximately 45 minutes, the reaction was quenched by addingsolid sodium tetrathionate to a final concentration of 5 mM.

D. Purification of conjugate. For small scale reactions, monosubstitutedconjugate was obtained using HPLC Zorbax (preparative) size exclusionchromatography. The desired monosubstituted conjugate product eluted at14.0-14.5 min (3.0 ml/min flow rate), while unreacted NR-LU-10 eluted at14.5-15 min and unreacted derivitized streptavidin eluted at 19-20 min.

For larger scale conjugation reactions, monosubstituted adduct isisolatable using DEAE ion exchange chromatography. After concentrationof the crude conjugate mixture, free streptavidin was removed therefromby eluting the column with 2.5% xylitol in sodium borate buffer, pH 8.6.The bound unreacted antibody and desired conjugate were thensequentially eluted from the column using an increasing salt gradient in20 mM diethanolamine adjusted to pH 8.6 with sodium hydroxide.

E. Characterization of conjugate.

1. HPLC size exclusion was conducted as described above with respect tosmall scale purification.

2. SDS-PAGE analysis was performed using 5% polyacrylamide gels undernon-denaturing conditions. Conjugates to be evaluated were not boiled insample buffer containing SDS to avoid dissociation of streptavidin intoits 15 kD subunits. Two product bands were observed on the gel, whichcorrespond to the mono- and di-substituted conjugates.

3. Immunoreactivity was assessed, for example, by competitive bindingELISA as compared to free antibody. Values obtained were within 10% ofthose for the free antibody.

4. Biotin binding capacity was assessed, for example, by titrating aknown quantity of conjugate with p-(I-125!iodobenzoylbiocytin.Saturation of the biotin binding sites was observed upon addition of 4equivalences of the labeled biocytin.

5. In vivo studies are useful to characterize the reaction product,which studies include, for example, serum clearance profiles, ability ofthe conjugate to target antigen-positive tumors, tumor retention of theconjugate over time and the ability of a biotinylated molecule to bindstreptavidin conjugate at the tumor. These data facilitate determinationthat the synthesis resulted in the formation of a 1:1streptavidin-NR-LU-10 whole antibody conjugate that exhibits bloodclearance properties similar to native NR-LU-10 whole antibody, andtumor uptake and retention properties at least equal to native NR-LU-10.

For example, FIG. 3 depicts the tumor uptake profile of theNR-LU-10-streptavidin conjugate (LU-10-StrAv) in comparison to a controlprofile of native NR-LU-10 whole antibody. LU-10-StrAv was radiolabeledon the streptavidin component only, giving a clear indication thatLU-10-StrAv localizes to target cells as efficiently as NR-LU-10 wholeantibody itself.

EXAMPLE XII Two-Step Pretargeting In Vivo

A ¹⁸⁶ Re-chelate-biotin conjugate (Re-BT) of Example I (MW≈1000;specific activity=1-2 mCi/mg) and a biotin-iodine-131 small molecule,PIP-Biocytin (PIP-BT, MW approximately equal to 602; specificactivity=0.5-1.0 mCi/mg), as discussed in Example VII above, wereexamined in a three-step pretargeting protocol in an animal model, asdescribed in Example V above. Like RE-BT, PIP-BT has the ability to bindwell to avidin and is rapidly cleared from the blood, with a serumhalf-life of about 5 minutes. Equivalent results were observed for bothmolecules in the two-step pretargeting experiments described herein.

NR-LU-10 antibody (MW≈150 kD) was conjugated to streptavidin (MW≈66 kD)(as described in Example XI above) and radiolabeled with ¹²⁵ I/PIP-NHS(as described for radioiodination of NR-LU-10 in Example IV.A., above).The experimental protocol was as follows:

    ______________________________________    Time 0        inject (i.v.) 200 μg NR-LU-10-StrAv                  conjugate;    Time 24-48 h  inject (i.v.) 60-70 fold                  molar excess of radiolabeled                  biotinyl molecule;    ______________________________________

and perform biodistributions at 2, 6, 24, 72, 120 hours after injectionof radiolabeled biotinyl molecule

NR-LU-10-streptavidin has shown very consistent patterns of bloodclearance and tumor uptake in the LS-180 animal model. A representativeprofile is shown in FIG. 4. When either PIP-BT or RE-BT is administeredafter allowing the LU-10-StrAv conjugate to localize to target cellsites for at least 24 hours, the tumor uptake of therapeuticradionuclide is high in both absolute amount and rapidity. For PIP-BTadministered at 37 hours following LU-10-StrAv (I-125) administration,tumor uptake was above 500 pMOL/G at the 40 hour time point and peakedat about 700 pMOL/G at 45 hours post-LU-10-StrAv administration.

This almost instantaneous uptake of a small molecule therapeutic intotumor in stoichiometric amounts comparable to the antibody targetingmoiety facilitates utilization of the therapeutic radionuclide at itshighest specific activity. Also, the rapid clearance of radionuclidethat is not bound to LU-10-StrAv conjugate permits an increasedtargeting ratio (tumor:blood) by eliminating the slow tumor accretionphase observed with directly labeled antibody conjugates. The pattern ofradionuclide tumor retention is that of whole antibody, which is verypersistent.

Experimentation using the two-step pretargeting approach andprogressively lower molar doses of radiolabeled biotinyl molecule wasalso conducted. Uptake values of about 20% ID/G were achieved atno-carrier added (high specific activity) doses of radiolabeled biotinylmolecules. At less than saturating doses, circulating LU-10-StrAv wasobserved to bind significant amounts of administered radiolabeledbiotinyl molecule in the blood compartment.

EXAMPLE XIII Asialoorosomucoid Clearing Agent and Two-Step Pretargeting

In order to maximize the targeting ratio (tumor:blood), clearing agentswere sought that are capable of clearing the blood pool of targetingmoiety-anti-ligand conjugate (e.g., LU-10-StrAv), without compromisingthe ligand binding capacity thereof at the target sites. One such agent,biotinylated asialoorosomucoid, which employs the avidin-biotininteraction to conjugate to circulating LU-10-StrAv, was tested.

A. Derivitization of orosomucoid. 10 mg human orosomucoid (Sigma N-9885)was dissolved in 3.5 ml of pH 5.5 0.1 M sodium acetate buffer containing160 mH NaCl. 70 μl of a 2% (w/v) CaCl solution in deionized (D.I.) waterwas added and 11 μl of neuraminidase (Sigma N-7885), 4.6 U/ml, wasadded. The mixture was incubated at 37° C. for 2 hours, and the entiresample was exchanged over a Centricon-10® ultrafiltration device(available from Amicon, Danvers, Mass.) with 2 volumes of PBS. Theasialoorosomucoid and orosomucoid starting material were radiolabeledwith I-125 using PIP technology, as described in Example IV above.

The two radiolabeled preparations were injected i.v. into female BALB/cmice (20-25 g), and blood clearance was assessed by serial retro-orbitaleye bleeding of each group of three mice at 5, 10, 15 and 30 minutes, aswell as at 1, 2 and 4 hours post-administration. The results of thisexperiment are shown in FIG. 5, with asialoorosomucoid clearing morerapidly than its orosomucoid counterpart.

In addition, two animals receiving each compound were sacrificed at 5minutes post-administration and limited biodistributions were performed.These results are shown in FIG. 6. The most striking aspects of thesedata are the differences in blood levels (78% for orosomucoid and 0.4%for asialoorosomucoid) and the specificity of uptake ofasialoorosomucoid in the liver (86%), as opposed to other tissues.

B. Biotinylation of asialoorosomucoid clearing agent and orosomucoidcontrol. 100 μl of 0.2 M sodium carbonate buffer, pH 9.2, was added to 2mg (in 1.00 ml PBS) of PIP-125-labeled orosomucoid and to 2 mgPIP-125-labeled asialoorosomucoid. 60 μl of a 1.85 mg/ml solution ofNHS-amino caproate biotin in DMSO was then added to each compound. Thereaction mixtures were vortexed and allowed to sit at room temperaturefor 45 minutes. The material was purified by size exclusion columnchromatography (PD-10, Pharmacia) and eluted with PBS. 1.2 ml fractionswere taken, with fractions 4 and 5 containing the majority of theapplied radioactivity (>95%). Streptavidin-agarose beads (Sigma S-1638)or -pellets were washed with PBS; and 20 μg of each biotinylated,radiolabeled protein was added to 400 μl of beads and 400 μl of PBS;vortexed for 20 seconds and centrifuged at 14,000 rpm for 5 minutes. Thesupernatant was removed and the pellets were washed with 400 μl PBS.This wash procedure was repeated twice more, and the combinedsupernatants were assayed by placing them in a dosimeter versus theirrespective pellets. The values are shown below in Table 4.

                  TABLE 4    ______________________________________    Compound         Supernatant                               Pellet    ______________________________________    orosomucoid      90%       10%    biotin-oroso     7.7%      92.%    asialoorosomucoid                     92%       8.0%    biotin-asialo    10%       90%    ______________________________________

C. Protein-Streptavidin Binding in vivo. Biotin-asialoorosomucoid wasevaluated for the ability to couple with circulating LU-10-StrAvconjugate in vivo and to remove it from the blood. Female BALB/c mice(20-25 g) were injected i.v. with 200 μg LU-10-StrAv conjugate. Clearingagent (200 μl PBS--group 1; 400 μg non-biotinylatedasialoorosomucoid--group 2; 400 μg biotinylated asialoorosomucoid--group3, and 200 μg biotinylated asialoorosomucoid--group 4) was administeredat 25 hours following conjugate administration. A fifth group receivedPIP-I-131-LU-10-StrAv conjugate which had been saturated prior toinjection with biotin--group 5. The 400 μg dose constituted a 10:1 molarexcess of clearing agent over the initial dose of LU-10-StrAv conjugate,while the 200 μg dose constituted a 5:1 molar excess. The saturatedPIP-I-131-LU-10-StrAv conjugate was produced by addition of a 10-foldmolar excess of D-biotin to 2 mg of LU-10-StrAv followed by sizeexclusion purification on a G-25 PD-10 column.

Three mice from each group were serially bled, as described above, at0.17, 1, 4 and 25 hours (pre-injection of clearing agent), as well as at27, 28, 47, 70 and 90 hours. Two additional animals from each group weresacrificed at 2 hours post-clearing agent administration and limitedbiodistributions were performed.

The blood clearance data are shown in FIG. 7. These data indicate thatcirculating LU-10-StrAv radioactivity in groups 3 and 4 was rapidly andsignificantly reduced, in comparison to those values obtained in thecontrol groups 1, 2 and 5. Absolute reduction in circulatingantibody-streptavidin conjugate was approximately 75% when compared tocontrols.

Biodistribution data are shown in tabular form in FIG. 8. Thebiodistribution data show reduced levels of conjugate for groups 3 and 4in all tissues except the liver, kidney and intestine, which isconsistent with the processing and excretion of radiolabel associatedwith the conjugate after complexation with biotinylatedasialoorosomucoid.

Furthermore; residual circulating conjugate was obtained from serumsamples by cardiac puncture (with the assays conducted in serum+PBS) andanalyzed for the ability to bind biotin (immobilized biotin on agarosebeads), an indicator of functional streptavidin remaining in the serum.Group 1 animal serum showed conjugate radiolabel bound about 80% toimmobilized biotin. Correcting the residual circulating radiolabelvalues by multiplying the remaining percent injected dose (at 2 hoursafter clearing agent administration) by the remaining percent able tobind immobilize biotin (the amount of remaining functional conjugate)leads to the graph shown in FIG. 9. Administration of 200 mgbiotinylated asialoorosomucoid resulted in a 50-fold reduction in serumbiotin-binding capacity and, in preliminary studies in tumored animals,has not exhibited cross-linking and removal of prelocalized LU-10-StrAvconjugate from the tumor. Removal of circulating targetingmoiety-anti-ligand without diminishing biotin-binding capacity at targetcell sites, coupled with an increased radiation dose to the tumorresulting from an increase in the amount of targeting moiety-anti-ligandadministered, results in both increased absolute rad dose to tumor anddiminished toxicity to non-tumor cells, compared to what is currentlyachievable using conventional radioimmunotherapy.

A subsequent experiment was executed to evaluate lower doses ofasialoorosomucoid-biotin. In the same animal model, doses of 50, 20 and10 μg asialoorosomucoid-biotin were injected at 24 hours followingadministration of the LU-10-StrAv conjugate. Data from animals seriallybled are shown in FIG. 10, and data from animals sacrificed two hoursafter clearing agent administration are shown in FIGS. 11A (bloodclearance) and 11B (serum biotin-binding), respectively. Doses of 50 and20 μg asialoorosomucoid-biotin effectively reduced circulatingLU-10-StrAv conjugate levels by about 65% (FIG. 11A) and, aftercorrection for binding to immobilized biotin, left only 3% of theinjected dose in circulation that possessed biotin-binding capacity,compared with about 25% of the injected dose in control animals (FIG.11B). Even at low doses (approaching 1:1 stoichiometry with circulatingLU-10-StrAv conjugate), asialoorosomucoid-biotin was highly effective atreducing blood levels of circulating streptavidin-containing conjugateby an in vivo complexation that was dependent upon biotin-avidininteraction.

EXAMPLE XIV Streptavidin Anti-Ligand in Tumors

A set of female nude mice, implanted subcutaneously with LS-180 humancolon carcinoma xenografts as described above, were randomized intogroups of 4 animals/timepoint. The mice were intravenously injected with200 μg of 1:1 mol/mol NR-LU-10 monoclonal antibody covalently coupled tostreptavidin (MAB-STRPT), with the conjugate formed as described inExample XI above. The streptavidin portion of the conjugate wasradiolabeled with paraiodophenyl (PIP) I-125, as described in Example IVabove. Groups of mice were sacrificed at 26, 30, 48, 96 and 144 hourspost-conjugate injection. Tissues were isolated, weighed and countedwith respect to iodine radionuclide content using conventionalprocedures therefor.

A second set of female nude mice bearing LS-180 xenografts were alsorandomized into groups of 4 animals/timepoint. These mice wereintravenously injected with 50 μg of NR-LU-10 monoclonal antibodyradiolabeled with paraiodophenyl (PIP) I-131 (MAB), as described inExample IV above. Mice were sacrificed at 4, 24, 48, 128 and 168 hourspost-radiolabeled monoclonal antibody injection. Tissues were isolated,weighed and counted with respect to iodine radionuclide content usingconventional procedures therefor.

For each data set, a radioactivity standard of the injected dose wasalso counted, and data were reduced to a percent of the total injecteddose per gram of tissue. FIG. 12 shows the percent injected dose/gram ofNR-LU-10-streptavidin-PIP-I-125 and N-RLU-10-PIP-I-131 in LS-180 tumorsover time. The NR-LU-10-streptavidin conjugate exhibits higher tumoruptake and a longer retention time as compared to NR-LU-10 alone.

EXAMPLE XV Streptavidin Ant-Ligand in Liver

Female nude mice xenografted with LS-180 tumor cells, as discussedabove, were randomized into groups of 4 animals/timepoint. Mice wereintravenously injected with 50 μg of biotinylated NR-LU-10 monoclonalantibody that was non-covalently coupled (to form a complex) throughbiotin-streptavidin binding to 30 μg of streptavidin. Prior tocomplexation in vivo, the antibody portion of the complex wasradiolabeled with I-125 using chloramine-T, and the streptavidin portionwas labeled with paraiodophenyl (PIP) I-131, both of the labelingprocedures having been described above. Mice were sacrificed at 4, 24,48, 96 and 144 hours post-conjugate injection. Tissues were isolated,weighed and counted with respect to the content of each iodineradionuclide using conventional procedures therefor.

A radioactivity standard of the injected doses of each complex componentwas also counted, and data were reduced to a percent of the totalinjected dose per gram of tissue. FIG. 13 shows the percent injecteddose per gram of streptavidin-PIP-I-131 (STREPT) andNR-LU-10-biotin-Chloramine-T-I-125 (MAB-BT) in liver over time. Thecomplex localized at the liver as a single molecule; however, theprocessing of the individual components thereof differed in the liver.The I-131-streptavidin label showed prolonged residence in the liver,while the monoclonal antibody label (I-125) was rapidly lost.

In another liver study, female nude mice xenografted with xenograftedwith LS-180 tumor cells, as discussed above, and were intravenouslyinjected with 200 μg of 1:1 mol/mol NR-LU-10 monoclonal antibodycovalently coupled to streptavidin, prepared as described in Example XIabove. The antibody portion of the conjugate was radiolabeled withparaiodophenyl (PIP-I-125). Twenty four hours later, the mice receivedan injection of 0.5 μg of paraiodophenyl (PIP I-131) biocytin. Mice weresacrificed at 28, 48, 120 and 168 hours post-conjugate injection.Tissues were isolated, weighed and counted with respect to the contentof each iodine radionuclide using conventional procedures therefor.

A radioactivity standard of the injected doses of each complex componentwas also counted, and data were reduced to a percent of the totalinjected dose per gram of tissue (%ID/G). FIG. 14 shows the percentinjected dose per gram of streptavidin-monoclonal antibody-PIP-I-125(STREP-MAB-I-125) and biocytin-PIP-I-131 (BT-I-131) in liver over time.When biocytin-PIP-I-131 was subsequently administered, the retention ofstreptavidin-bound biotin radiolabel (I-131) was prolonged relative tothe retention of the antibody-bound label (I-125) on the same moiety inthe liver.

EXAMPLE XVI Tumor Uptake of PIP-Biocytin

PIP-Biocytin, as prepared and described in Example VII above, was testedto determine the fate thereof in vivo. The following data are based onexperimentation with tumored nude mice (100 mg LS-180 tumor xenograftsimplanted subcutaneously 7 days prior to study) that received, at time0, 200 μg of I-125 labeled NR-LU-10-Streptavidin conjugate (950 pmol),as discussed in Example XI above. At 24 hours, the mice received an i.v.injection of PIP-I-131-biocytin (40 μCi) and an amount of cold carrierPIP-I-127 biocytin corresponding to doses of 42 μg (69,767 pmol), 21 μg(34,884 pmol), 5.7 μg (9468 pmol), 2.85 μg (4734 pmol) or 0.5 μg (830pmol). Tumors were excised and counted for radioactivity 4 hours afterPIP-biocytin injection, and the tumor uptake data are shown in FIGS. 15A(%ID/G v. Dose) and 15B (pMOL/G v. Dose).

The three highest doses produced PIP-biocytin tumor localizations ofabout 600 pmol/g. Histology conducted on tissues receiving the twohighest doses indicated that saturation of tumor-bound streptavidin wasachieved. Equivalent tumor localization observed at the 5.7 μg dose(FIG. 15B) is indicative of streptavidin saturation as well. Incontrast, the two lowest doses produced lower absolute tumorlocalization of PIP-biocytin, despite equivalent localization ofNR-LU-10-Streptavidin conjugate (tumors in all groups averaged about 40%ID/G for the conjugate).

The lowest dose group (0.5 μg) exhibited high efficiency tumor deliveryof PIP-I-131-biocytin, which efficiency increased over time, as shown inFIG. 16A. A peak uptake of 85.0% ID/G was observed at the 120 hour timepoint (96 hours after administration of PIP-biocytin). Also, theabsolute amount of PIP-biocytin, in terms of % ID, showed a continualincrease in the tumor over all of the sampled time points (FIG. 16B).The decrease in uptake on a % ID/G basis (FIG. 16A) at the 168 hour timepoint resulted from significant growth of the tumors between the 120 and168 hour time points.

In addition, FIG. 17A shows the co-localization of NR-LU-10-Streptavidinconjugate (LU-10-StrAv) and the subsequently administered PIP-Biocytinat the same tumors over time. The localization of radioactivity attumors by PIP-biocytin exhibited a pattern of uptake and retention thatdiffered from that of the antibody-streptavidin conjugate (LU-10-StrAv).LU-10-StrAv exhibited a characteristic tumor uptake pattern that isequivalent to historical studies of native NR-LU-10 antibody, reaching apeak value of 40% ID/G between 24 and 48 hours after administration. Incontrast, the PIP-Biodytin exhibited an initial rapid accretion in thetumor, reaching levels greater than those of LU-10-StrAv by 24-hoursafter PIP-Biocytin administration. Moreover, the localization ofPIP-Biocytin continued to increase out to 96 hours, when theconcentration of radioactivity associated with the conjugate has begunto decrease. The slightly greater amounts of circulating PIP-Biocytincompared to LU-10-StrAv at these time points (shown in FIG. 17B)appeared insufficient to account for this phenomenon.

As FIG. 18 clearly shows, the ratio of PIP-Biocytin to LU-10-StrAv inthe tumor increased continually during the experiment, while the ratioin the blood decreased continually. This observation is consistent witha process involving continual binding of targeting moiety-containingconjugate (with PIP-Biocytin bound to it) from the blood to the tumor,with subsequent differential processing of the PIP-Biocytin and theconjugate. Since radiolabel associated with the streptavidin conjugatecomponent (compared to radiolabel associated with the targeting moiety)has shown increased retention in organs of metabolic processing(Examples XIV and XV above), PIP-Biocytin associated with thestreptavidin appears to be selectively retained by the tumor cells.Because radiolabel is retained at target cell sites, a greateraccumulation of radioactivity at those sites results.

The AUC_(tumor) /AUC_(blood) for PIP-Biocytin is over twice that of theconjugate (4.27 compared to 1.95, where AUC means "area under thecurve"). Further, the absolute AUC_(tumor) for PIP-Biocytin is nearlytwice that of the conjugate (9220 compared to 4629). Consequently, anincrease in radiation dose to tumor was achieved.

Kits containing one or more of the components described above are alsocontemplated. For instance, radiohalogenated biotin may be provided in asterile container for use in pretargeting procedures. A chelate-biotinconjugate provided in a sterile container is suitable forradiometallation by the consumer; such kits would be particularlyamenable for use in pretargeting protocols. Alternatively,radiohalogenated biotin and a chelate-biotin conjugate may be vialed ina non-sterile condition for use as a research reagent.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. A clearing agent conjugate which comprises (i) atleast one binding moiety selected from the group consisting of avidin,streptavidin, analogs and derivatives of either binding biotin with highaffinity and further comprising (ii) a clearance directing moiety whichis proteinaceous which comprises exposed galactose residues, at leastsome of which have been introduced by synthetic methods, wherein saidclearing agent comprises a molecular weight ranging from about 40,000 toabout 200,000 daltons and which is capable of enhancing in vivoclearance of a second conjugate from the blood via Ashwell receptormediated clearance, wherein said second conjugate comprises thecomplementary binding partner of avidin or streptavidin, which iscontained in the clearing agent conjugate.
 2. A clearing agent conjugatewhich comprises (i) at least one binding moiety selected from the groupconsisting of avidin, streptavidin, derivatives and analogs of eitherbinding biotin with high affinity, and further comprises (ii) aclearance directing moiety which is proteinaceous, which comprises asufficient number of exposed galactose residues that the clearing agentprovides for Ashwell receptor mediated clearance of a second conjugatewhich comprises biotin.
 3. The clearing agent of claim 1, wherein theclearance-directing moiety is selected from the group consisting ofhuman serum albumin, non-immunogenic serum-soluble protein,polyglutamate, polyglycine, polyarginine, and polyaspartate.
 4. Theclearing agent of claim 1, wherein the clearance-directing moiety isselected from the group consisting of galactose-derivatized IgM, IgG,and transferrin.
 5. The clearing agent of 2, wherein theclearance-directing moiety is asialoorosomucoid, a derivative, or ananalog thereof.